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Laboratory Equipment

for
MAI NTENANCE
Manual
2nd Edi t i on
Please go to the Table of Contents to access additional chapters.
Laboratory Equipment
for
MAI NTENANCE
Manual
2nd Edi t i on
WHO Library Cataloguing-in-Publication Data
Maintenance manual for laboratory equipment, 2
nd
ed.
1.Laboratory equipment. 2.Maintenance. 3.Manuals. I.World Health Organization. II.Pan American Health Organization.
ISBN 978 92 4 159635 0 (NLM classication: WX 147)
World Health Organization 2008
All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland
(tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications whether for sale or for
noncommercial distribution should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; e-mail: permissions@who.int).
The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health
Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on
maps represent approximate border lines for which there may not yet be full agreement.
The mention of specic companies or of certain manufacturers products does not imply that they are endorsed or recommended by the World Health Organization in
preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital
letters.
All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being
distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the
World Health Organization be liable for damages arising from its use.
Design and Layout: LIV Com Srl, Morges Switzerland
Printed in Spain
Contact:
Dr G. Vercauteren, Coordinator, Diagnostics and Laboratory Technology, Department of Essential Health Technologies, World Health Organization, 20 Avenue Appia, 1211 Geneva
2, Switzerland
This document is available at www.who.int/diagnostics_laboratory
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
iii
TABLE OF FIGURES viii
ACKNOWLEDGEMENTS x
INTRODUCTION xi
CHAPTER 1 MICROPLATE READER 1
Photograph of microplate reader 1
Purpose of the microplate reader 1
Operation principles 1
Installation requirements 3
Routine maintenance 3
Troubleshooting table 4
Basic defnitions 5
CHAPTER 2 MICROPLATE WASHER 7
Photograph of microplate washer 7
Purpose of the microplate washer 7
Operation principles 7
Installation requirements 9
Routine maintenance 9
Troubleshooting table 11
Basic defnitions 12
CHAPTER 3 pH METER 13
Purpose of the equipment 13
Photograph and components of the pH meter 13
Operation principles 13
pH meter components 14
Typical circuit 15
Installation requirements 16
General calibration procedure 16
General maintenance of the pH meter 17
Basic maintenance of the electrode 18
Troubleshooting table 18
Basic defnitions 19
Annex: The pH theory 20
Table of Contents
TABLE OF CONTENTS
iv
CHAPTER 4 BALANCES 21
Photographs of balances 21
Purpose of the balance 22
Operation principles 22
Installation requirements 26
Routine maintenance 27
Troubleshooting table 28
Basic defnitions 29
CHAPTER 5 WATER BATH 31
Diagram of a water bath 31
Operation principles 31
Water bath controls 32
Water bath operation 32
Troubleshooting table 34
Basic defnitions 34
CHAPTER 6 BIOLOGICAL SAFETY CABINET 35
Illustration of a biological safety cabinet 35
Purposes of the equipment 35
Operation principles 35
Biological safety 39
Installation requirements 39
Using the safety cabinet 39
Routine maintenance 40
Functional evaluation (alternative) 41
Table of functional evaluation of biological safety cabinets 42
Troubleshooting table 43
Basic defnitions 44
CHAPTER 7 CENTRIFUGE 45
Photograph of centrifuge 45
Purpose of the centrifuge 45
Operation principles 45
Components of the centrifuge 46
Installation requirements 48
Routine maintenance 48
Appropriate management and storage recommendations 48
Troubleshooting table 50
Basic defnitions 52
CHAPTER 8 WATER DISTILLER 53
Diagram of a water distiller 53
Purpose of the water distiller 53
Operation principles 54
Installation requirements 54
Routine maintenance 55
Troubleshooting table 56
Basic defnitions 57
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CHAPTER 9 DILUTOR 59
Diagram of a dilutor 59
Purpose of the dilutor 59
Operation principles 60
Installation requirements 61
Routine maintenance 61
Troubleshooting table 63
Basic defnitions 64
CHAPTER 10 DISPENSER 65
Photograph and diagram of the dispenser 65
Purpose of the dispenser 65
Requirements for operation 67
Routine maintenance 67
Troubleshooting table 68
Basic defnitions 68
CHAPTER 11 SPECTROPHOTOMETER 69
Photograph of spectrophotometer 69
Purpose of the equipment 69
Operation principles 69
Spectrophotometer components 72
Installation requirements 73
Spectrophotometer maintenance 73
Good practices when using the spectrophotometer 75
Troubleshooting table 77
Basic defnitions 79
CHAPTER 12 AUTOCLAVE 81
Photograph of the autoclave 81
Purpose of the autoclave 81
Operation principles 82
Operation of the autoclave 84
Installation requirements 87
Routine maintenance 88
Maintenance of specialized components 90
Troubleshooting table 91
Basic defnitions 92
CHAPTER 13 DRYING OVEN 93
Photograph of drying oven 93
Purpose of the oven 93
Operating principles 93
Installation requirements 94
Oven operation 94
Oven controls 95
Quality control 96
Routine maintenance 96
Troubleshooting table 97
Basic defnitions 98
TABLE OF CONTENTS
vi
CHAPTER 14 INCUBATOR 99
Photograph of incubator 99
Operating principles 99
Incubator controls 101
Installation requirements 101
Routine maintenance and use of the incubator 101
Troubleshooting table 103
Basic defnitions 104
CHAPTER 15 MICROSCOPE 105
Photographs of microscopes 105
Purpose of the equipment 106
Operation principles 106
Installation requirements 108
Description of potential problems with microscopes 109
General maintenance of the microscope 111
Troubleshooting table 115
Basic defnitions 116
CHAPTER 16 PIPETTES 119
Photographs of pipettes 119
Purpose of the pipettes 120
Operation principles of the pipette 120
Requirements for use 120
Using the pipette 121
Routine maintenance 122
Troubleshooting table 125
Basic defnitions 126
CHAPTER 17 STIRRING HEATING PLATE 127
Photograph of the stirring heating plate 127
Operation principles 127
Controls of the stirring heating plate 127
Installation requirements 128
Operation of the stirring heating plate 128
Routine maintenance 128
Troubleshooting table 129
Basic defnitions 129
CHAPTER 18 REFRIGERATORS AND FREEZERS 131
Photograph of a refrigerated storage unit 131
Purpose of refrigerated storage units 132
Operation principles 132
Installation requirements 133
Refrigerator control circuit 134
Refrigerator operation 134
Refrigerator routine maintenance 135
Troubleshooting table 137
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
vii
Operation of ultralow freezers 138
Turning the unit on 138
Routine maintenance 139
Troubleshooting table 140
Basic defnitions 141
CHAPTER 19 CHEMISTRY ANALYSERS 143
Photographs of chemistry analysers 143
Purpose of chemistry analysers 144
Operation principle 144
Components 144
Installation requirements 145
Operation of the dry chemistry analyser 145
Operation of the wet chemistry analyser 146
Routine maintenance of chemistry analysers 146
Non-routine maintenance and troubleshooting 147
Troubleshooting table 148
Basic defnitions 148
CHAPTER 20 COLORIMETERS 149
Photograph of colorimeter 149
Purpose of the colorimeter 149
Operating principle 149
Components 150
Installation requirements 150
Operation of the colorimeter 150
Operation of the haemoglobinometer 151
Routine maintenance 151
Troubleshooting table 154
Basic defnitions 155
BIBLIOGRAPHY 157
TABLE OF FI GURES
viii
Table of Figures
Figure 1 Equipment used for ELISA tests 2
Figure 2 Microplate washer 8
Figure 3 Well profles 8
Figure 4 Diagram of a pH meter 14
Figure 5 Types of electrodes 15
Figure 6 Example of a typical pH meter control circuit 15
Figure 7 Spring balance 22
Figure 8 Sliding weight scale 22
Figure 9 Analytical balance 22
Figure 10 Upper plate balance 23
Figure 11 Substitution balance 23
Figure 12 Components of the electronic balance 24
Figure 13 Compensation force principle 24
Figure 14 Classifcation of balances by exactitude 25
Figure 15 Analytical balance control panel 26
Figure 16 Water bath 31
Figure 17 Immersion and external resistors 31
Figure 18 Water bath controls 32
Figure 19 Biological safety cabinet 35
Figure 20 Centrifugal force concept 46
Figure 21 Water distiller 53
Figure 22 Dilutor diagram 59
Figure 23 Dilutor controls 60
Figure 24 Syringe and dispenser 61
Figure 25 Dispenser 65
Figure 26 Dispenser and accessories 66
Figure 27 Interaction of light with matter 70
Figure 28 Absorbance phenomenon 71
Figure 29 Spectrophotometer components 72
Figure 30 Refraction of light 79
Figure 31 Difraction grid 80
Figure 32 Vapour circuit of an autoclave 83
Figure 33 Space required for autoclave 87
Figure 34 Compressed air connection 87
Figure 35 Vapour connection 88
Figure 36 Vapour generator 89
Figure 37 Electronic control of the oven 95
Figure 38 Electrical circuit of the oven 95
Figure 39 Heat transfer systems used in incubators 100
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
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Figure 40 Incubator controls 101
Figure 41 Positive (convergent) lens 106
Figure 42 Optics of the convergent lens 106
Figure 43 Diagram of a microscope 107
Figure 44 Cross-section of a microscope 108
Figure 45 Binocular head 109
Figure 46 Lighting system 109
Figure 47 Platform, plate or mechanical stage 110
Figure 48 Revolving, objective holder 110
Figure 49 Body of the microscope 111
Figure 50 Diagram of a pipette 119
Figure 51 Types of pipettes 120
Figure 52 Phases of pipette use 121
Figure 53 Disassembly of a pipette 123
Figure 54 Stirring heating plate controls 127
Figure 55 Induction motor 129
Figure 56 Refrigeration circuit 132
Figure 57 Control circuit of the refrigerator 134
Figure 58 Blood bank refrigerator controls 135
Figure 59 Ultralow temperature freezer control 138
Figure 60 Basic diagram of refectance photometry on a test strip 144
Figure 61 Ulbrichts sphere 145
Figure 62 Basic components of a photometer 145
Figure 63 Controls of a portable colorimeter 150
PREFACE
x
Acknowledgements
This manual is a revised edition of Manual de mantenimiento para equipo de laboratorio (PAHO, 2005) translated from
Spanish into English. Revisions include additional chapters on laboratory equipment commonly used in some laboratories
and updates allowing global use of the manual.
The revised version has been prepared under the direction of Dr Gaby Vercauteren, World Health Organization, Geneva,
Switzerland and in coordination with Dr Jean-Marc Gabastou, Pan-American Health Organization/World Health Organization,
Washington, DC, USA; translated by Ms Christine Philips; reviewed by Ms Mercedes Prez Gonzlez and adapted, revised and
edited by Mrs Isabelle Prudhomme.
WHO kindly expresses thanks to those who have participated at all levels in the elaboration of this manual. WHO wishes to
acknowledge the original contribution of Dr Jorge Enrique Villamil who wrote the frst edition of this manual in 2005 (Manual
para mantenimiento de equipo de laboratorio, ISBN 92 75 32590 1) and Dr Jean-Marc Gabastou and Mr Antonio Hernndez,
Reviewers at Essential Medicines Vaccines and Health Technologies at PAHO.
WHO also thanks manufacturers who have granted permission to use their images in this publication.
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
xi
Introduction
This manual has been developed to support personnel employed in health laboratories. Its purpose is to give a better
understanding of the technical requirements regarding installation, use and maintenance of various types of equipment which
play an important role in performing diagnostic testing. The manual also aims to provide support to personnel responsible
for technical management, implementation of quality management and maintenance.
Due to the diversity of origins, brands and models, this manual ofers general recommendations. Equipment-specifc details
are explained in depth in the maintenance and installation user manuals from manufacturers. These should be requested
and ordered through the procurement processes of the individual agencies and professionals responsible for the acquisition
of technology, or directly from the manufacturer.
This manual was originally developed by the Pan-American Health Organization (PAHO) to support improved quality
programmes which PAHO promotes in regional laboratories. The English version was produced by WHO to further expand
support for quality programmes in other regions. The revised edition now includes 20 equipment groups selected to cover
those most commonly used in low to medium technical complexity laboratories across the world. Given the diferences in
technical complexity, brands and existing models, each chapter has been developed with basic equipment in mind, including
new technology where relevant. The following information is included in each chapter:
Groups of equipment, organized by their generic names. Alternative names have also been included.
Photographs or diagrams, or a combination of both to identify the type of equipment under consideration.
A brief explanation on the main uses or applications of the equipment in the laboratory.
A basic description of the principles by which the equipment operates with explanations of principles or physical and/or
chemical laws which the interested reader can or should study in depth.
Installation requirements with emphasis on the electrical aspects and the requirements for safe installation and operation,
including worldwide electrical standards.
Basic routine maintenance, classifed according to the required frequency (daily, weekly, monthly, quarterly, annually
or sporadically). The procedures are numbered and presented in the actual sequence in which these should take place
(model-specifc procedures can be found in the manuals published by the manufacturers).
Troubleshooting tables with the most frequent problems afecting the equipment with possible causes and actions that
may resolve these problems.
A list of basic defnitions of some of the specialized terms used.
For some equipment, additional themes related to calibration, quality control and design (with operational controls).
This information, along with good use and care, helps to maintain laboratory equipment in optimal condition.
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
1
The microplate reader also known as Photometric
micro-plate reader or ELISA reader is a specialized
spectrophotometer designed to read results of the ELISA
test, a technique used to determine the presence of
antibodies or specifc antigens in samples. The technique
is based on the detection of an antigen or antibodies
captured on a solid surface using direct or secondary,
labelled antibodies, producing a reaction whose product
can be read by the spectrophotometer. The word ELISA is
the acronym for Enzyme-Linked Immunosorbent Assay.
This chapter covers the use of microplate readers for ELISA
testing. For additional information on the instrument
principles of operation and maintenance, consult Chapter
11 discussing the spectrophotometer.
PHOTOGRAPH OF MICROPLATE READER
PURPOSE OF THE MICROPLATE READER
The microplate reader is used for reading the results of ELISA
tests. This technique has a direct application in immunology
and serology. Among other applications it confrms the
presence of antibodies or antigens of an infectious agent in
an organism, antibodies from a vaccine or auto-antibodies,
for example in rheumatoid arthritis.
OPERATION PRINCIPLES
The microplate reader is a specialized spectrophotometer.
Unlike the conventional spectrophotometer which facilitates
readings on a wide range of wavelengths, the microplate
reader has filters or diffraction gratings that limit the
wavelength range to that used in ELISA, generally between
400 to 750 nm (nanometres). Some readers operate in the
ultraviolet range and carry out analyses between 340 to 700
nm. The optical system exploited by many manufacturers
uses optic fbres to supply light to the microplate wells
containing the samples. The light beam, passing through
the sample has a diameter ranging between 1 to 3 mm.
A detection system detects the light coming from the
sample, amplifes the signal and determines the samples
absorbance. A reading system converts it into data allowing
the test result interpretation. Some microplate readers use
double beam light systems.
Test samples are located in specially designed plates with
a specifc number of wells where the procedure or test is
carried out. Plates of 8 columns by 12 rows with a total of
96 wells are common. There are also plates with a greater
number of wells. For specialized applications, the current
trend is to increase the number of wells (384-well plates)
to reduce the amount of reagents and samples used and a
greater throughput. The location of the optical sensors of the
microplate reader varies depending on the manufacturers:
these can be located above the sample plate, or directly
underneath the plates wells.
Nowadays microplate readers have controls regulated by
microprocessors; connection interfaces to information
systems; quality and process control programs, which by
means of a computer, allow complete test automation.
Chapter 1
Microplate Reader
GMDN Code 37036
ECRI Code 16-979
Denomination Photometric micro-plate reader
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CHAPTER 1 MI CROPLATE READER
2
Equipment required for ELISA testing
In order to perform the ELISA technique, the following
equipment is required:
1. Microplate reader.
2. Microplate washer (Chapter 2).
3. Liquid dispensing system (multi-channel pipettes may
be used).
4. Incubator to incubate the plates.
Figure 1 illustrates how this equipment is interrelated.
Mechanical phases of the ELISA technique
Using the equipment
When an ELISA test is conducted, it typically follows these
steps:
1. A frst washing of the plate may be done using the
microplate washer.
2. Using a liquid dispenser or the multi-channel pipettes,
wells are flled with the solution prepared to be used in
the test.
3. The plate is placed in the incubator where at a controlled
temperature, a series of reactions take place.
Stages 1, 2 and 3 can be repeated several times depending
on the test, until the reagents added have completed their
reactions.
Finally, when all the incubation steps have been completed,
the plate is transferred to the microplate reader. The reading
of the plate is done and a diagnosis can be deduced.
Biochemical phases of the ELISA technique
1
The ELISA technique from a biochemical point of view:
1. The plate wells are coated with antibodies or antigens.
2. Samples, controls and standards are added to the wells
and incubated at temperatures ranging between room
temperature and 37 C for a determined period of
time, according to the tests characteristics. During the
incubation, the samples antigen binds to the antibody
coated to the plate; or the antibody in the sample binds
to the antigen coated on the plate, according to their
presence and quantity in the sample analyzed.
3. After incubation, the unbound antigen or antibodies are
washed and removed from the plate by the microplate
washer using an appropriate washing bufer.
4. Next, a secondary antibody, called the conjugate, is
added. This harbours an enzyme which will react with
a substrate to produce a change of colour at a later
step.
5. Then begins a second period of incubation during
which this conjugate will bind to the antigen-antibody
complex in the wells.
6. After the incubation, a new washing cycle is done to
remove unbound conjugate from the wells.
7. A substrate is added. The enzyme reacts with the
substrate and causes the solution to change in colour.
This will indicate how much antigen-antibody complex
is present at the end of the test.
8. Once the incubation time is completed, a reagent
is added to stop the enzyme-substrate reaction and
to prevent further changes in colour. This reagent is
generally a diluted acid.
9. Finally, the plate in is read by the microplate. The
resulting values are used to determine the specific
amounts or the presence of antigens or antibodies in
the sample.
Note: Some of the wells are used for
standards and controls. Standards allow
the cut-of points to be defned. The
standards and controls are of known
quantities and are used for measuring
the success of the test, evaluating data
against known concentrations for each
control. The process described above
is common, although there are many
ELISA tests with test-specifc variants.
1
More detailed explanations must be consulted in
specialized literature.



Dispensing
System
ELISA Plate
Washer
Incubator
ELISA
Reader
Computer
Figure 1. Equipment used in ELISA tests
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
3
INSTALLATION REQUIREMENTS
In order for the microplate reader to operate correctly, the
following points need to be respected:
1. A clean, dust free environment.
2. A stable work table away from equipment that vibrates
(centrifuges, agitators). It should be of a suitable size so
that there is working space at the side of the microplate
reader. The required complementary equipment for
conducting the technique described above is: washer,
incubator, dispenser and computer with its peripheral
attachments.
3. An electrical supply source, which complies with the
countrys norms and standards. In the countries of the
Americas for example, 110 V and 60 Hertz frequencies
are generally used, whereas other regions of the World
use 220-240V, 50/60HZ.
Calibration of the microplate reader
The calibration of a microplate reader is a specialized process
which must be executed by a technician or trained engineer
following the instructions provided by each manufacturer.
In order to do the calibration, it is necessary to have a set
of grey flters mounted on a plate of equal geometric size
to those used in the analyses. Manufacturers provide these
calibration plates for any wavelength the equipment uses.
Calibration plates are equipped with at least three pre-
established optic density values within the measurement
ranges; low, medium, and high value. In order to perform
the calibration, follow this process:
1. Place the calibration plate on the equipment.
2. Carry out a complete reading with the calibration plate.
Verify if there are diferences in the readings obtained
from well to well. If this is the case, invert the plate (180)
and repeat the reading to rule out that diferences are
attributed to the plate itself. In general, it is accepted
that the instrument does not need further calibration if
the plate results are as expected at two wavelengths.
3. Verify if the reader requires calibration. If so, proceed
with the calibration following the routine outlined by
the manufacturer, verifying that the readings linearity
is maintained as rigorously as possible.
4. If the instrument does not have a calibration plate, verify
it by placing a coloured solution in the wells of a plate
and immediately carry out a complete reading. Then
invert the plate 180 and read the plate again. If both
readings display identical, average values in each row,
the reader is calibrated.
5. Verify that the reader is calibrated, column by column.
Place a clean, empty plate and carry out a reading. If
there is no diference between each of the average
reading of the frst to the last column, it can be assumed
that the reader is calibrated.
ROUTINE MAINTENANCE
Maintenance described next focuses exclusively on the
microplate reader. The maintenance of the microplate
washer is described in Chapter 2.
Basic maintenance
Frequency: Daily
1. Review that optical sensors of each channel are clean.
If dirt is detected, clean the surface of the windows of
the light emitters and the sensors with a small brush.
2. Confrm that the lighting system is clean.
3. Verify that the readers calibration is adequate. When
the daily operations begin, let the reader warm up for
30 minutes. Next, do a blank reading and then read a
full plate of substrate. The readings must be identical.
If not, invert the plate and repeat the reading in order
to determine if the deviation originated in the plate or
the reader.
4. Examine the automatic drawer sliding system. It must
be smooth and constant.
Preventive maintenance
Frequency: Quarterly
1. Verify the stability of the lamp. Use the calibration plate,
conducting readings with intervals of 30 minutes with
the same plate. Compare readings. There must be no
diferences.
2. Clean the detectors optical systems and the lighting
systems.
3. Clean the plate drawer.
4. Verify the alignment of each well with the light emission
and detection systems.
CHAPTER 1 MI CROPLATE READER
4
TROUBLESHOOTING TABLE
PROBLEM PROBABLE CAUSE SOLUTION
The reader gives a reading that does not make sense. The illumination lamp is out of service. Replace the lamp with one with the same
characteristics as the original.
The readers readings vary from row to row. Dirty optical sensors. Clean the sensors.
The illumination systems lenses or parts are dirty. Clean the lighting systems lenses.
Lack of calibration in one or more channels. Verify the calibration of each one of the channels.
The reader displays high absorbance values. Reagents expired and/or incorrectly prepared. Check to see if the TMB is colourless and the
preparation adequate.
Contamination with other samples. Repeat the test verifying the labelling, the washer
and how the pipette was used.
Incorrect wavelength flter. Verify the recommended wavelength for the test.
Adjust if it is incorrect.
Insuf cient or inef cient washing. Verify the washing method used. Use an appropriate
quality control test.
Very long incubation time or very high temperature. Check incubation times and temperatures.
Incorrect sample dilution. Check process for sample dilution.
Some reagent was omitted. Verify that the test has been carried out according to
the established procedure.
The reader displays low absorbance values. Very short incubation time and very low
temperature.
Check temperatures and incubation times.
The reagents were not at room temperature. Check that the reagents are stable at room
temperature.
Excessive washing of the plate. Adjust the washing process to what the test
manufacturers indicate.
Incorrect wavelength flter. Verify the wavelength selected. Use wavelength
recommended for the test.
Expired or incorrectly prepared reagents. Check the used reagents. Test the dilutions.
A reagent was omitted. Verify that the test was done according to the
established procedure.
The plate displays scratches at the bottom of the
wells.
Prepare a new plate and repeat the test.
Incorrectly selected or dirty plate. Verify the type of plate used. Prepare a new plate
and repeat the test.
The plate wells have dried up. Change the manner in which the plate is washed.
The plate is incorrectly placed or is seated unevenly
in the reader.
Check the placement of the plate. Repeat the
reading.
Humidity or fngerprints on the outer part of the
bottom of the plate.
Verify that the plate under the bottom of the wells
is clean.
Residual quantities of washing bufer in the wells
before adding the substrate.
Confrm that the washing bufer is completely
removed.
The substrate tablets do not dissolve completely. Verify that the tablets dissolve correctly.
The substrate tablet has been contaminated by
humidity or metal clips or is not complete.
Test the integrity and handling of substrate tablets.
The position of the blank well could have been
changed and an incorrect quantity has been
subtracted at each reading.
Verify that the plate set-up is correct.
The reader displays unexpected variation in the
optical density readings.
The readers lamp is unstable. Replace the lamp with one that has similar
characteristics as the original.
The reader displays a gradual increase or decrease
from column to column.
Inappropriate calibration of the plates advance
motor.
Calibrate the advance so that at each step the wells
remain exactly aligned with the lighting system.
The optical density readings are very low compared
to the operators optical evaluation criteria.
The reading is being carried out with a diferent
wavelength than required for the test.
Verify the wavelength used when conducting
the reading. If this is the problem, adjust the
wavelength and repeat the reading. Verify that the
recommended wavelength flter has been selected.
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
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Low reproducibility. Sample homogeneity. Mix the reagents before use. Allow these to
equilibrate to room temperature.
Incorrect pipetting procedure. Ensure pipettes tips are changed between samples
and that excessive liquid inside is removed.
Reader not calibrated. Check the calibration. Use an appropriate quality
control set.
instrument.
Wait until the reader has warmed up to its operating
temperature.
Expired reagents. Verify the expiry dates of the reagents.
when washed.
The data are not transferred from the reader to the
microprocessor.
Verify selected codes.
transmission plugs.
manufacturer.
Misaligned light beam. The reader was transferred or moved without using
the necessary precautions.
Call the specialized service technician.
The light source lamp has been changed and
the replacement has not been installed or aligned
correctly.
Verify its assembly and alignment.
The plate was incorrectly loaded.
reading carrying out the adjustments.
the reader. reading carrying out the adjustments.
Computer fails to indicate the error codes. The programme which controls the activation of

not validated by the manufacturer.
Call the specialized service technician.
The blank sample shows high absorbance. Contaminated substrate. Check that TMB is colourless and its preparation.
The reader demonstrates failure in detecting errors. Various components of the system display failure,
such as the liquid level detection system.
Call the specialized service technician.
BASIC DEFINITIONS
Chemiluminescence. Emission of light or luminescence resulting directly from a chemical reaction at environmental temperatures.
ELISA (Enzyme-Linked Immunosorbent Assay). Biochemical technique used mainly in Immunology to detect the presence of an antibody or an antigen in a
sample.
ELISA plate. Consumable standardized to carry out the ELISA technique. Generally, plates have 96 wells in a typical confguration of 8 rows by 12 columns. There
are also ELISA plates with 384 wells or up to 1536 wells for specialized high throughput testing in centres with high demand.
Microplate washer. Equipment used for washing plates during specifc stages of an ELISA test with the aim of removing unbound components during reactions.
Microplate washers use special bufers in the washing process.
Enzyme. Protein that accelerates (catalyses) chemical reactions.
Fluorophore. Molecules absorbing light at a determined wavelength and emitting it at a higher wavelength.
Microplate reader. The name given to spectrophotometers with the capacity to read microplates.
TMB. Tetramethylbenzidine, a substrate for the horseradish peroxidase (HRP) enzyme.
alarms and warning messages is defective or is
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
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Chapter 2
Microplate Washer
The microplate washer or plate or ELISA washer is designed to
perform washing operations required in the ELISA technique.
The microplate washer performs the washing of the ELISA
plates wells during the diferent stages of the technique.
PHOTOGRAPH OF MICROPLATE WASHER
PURPOSE OF THE MICROPLATE WASHER
The microplate washer has been designed to supply cleaning
buffers required for the ELISA technique in a controlled
manner. In the same fashion, the equipment removes from
each well, substances in excess from the reaction. Depending
on the test performed, the washer can intervene from one
to four times, supplying the washing bufer, agitating and
removing the unbound reagents
1
until the programmed
times and cycles are completed. The washer has of two
reservoirs; one for the washing bufer, the other for the waste
generated during the washing process.
OPERATION PRINCIPLES
The microplate washer has been designed to perform
washing operations in the ELISA technique. The equipment
possesses at least, the following subsystems which vary
depending on the manufacturers design.
Control subsystem. Generally, the washer is controlled
by microprocessors allowing programming and
controlling steps to be performed by the washer such
as: number of washing cycles
2
(15); expected times;
supplying and extracting pressures; plate format
(96384 wells); suction function adjustment according
to the type of well
3
(fat bottom, V bottom or rounded
bottom or strips used); volumes distributed or aspirated;
the soaking and agitation cycles, etc.
Supply subsystem. In general, this comprises a reservoir
for the washing solution; one or several pumps; usually
a positive displacement type syringe and a dispenser
head that supplies the washing solution to the diferent
wells by means of needles. The head usually comes
with eight pairs of needles for washing and aspirating
simultaneously the wells of the same row (the supply
and extraction sub-systems converge on the head).
There are models with twelve pairs of needles and others
that conduct the washing process simultaneously in all
the wells. Some washers ofer the possibility of working
with diferent types of washing solutions, performing
the solution changes according to the program entered
by the operator.
1
See a brief explication of the ELISA technique in Chapter 1, Microplate
Reader.
2
The exact number of washing operations required depends on the assay
used. This is explained in each manufacturers test instruction manual.
3
If the bottom is fat, the suction needle is located very close to one of wells
faces; if it is rounded or V-shaped, the suction needle is centered.
GMDN Code 17489
ECRI Code 17-489
Denomination Micro-plate washer
P
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CHAPTER 2 MI CROPLATE WASHER
8
Extraction or suction system. This requires a vacuum
mechanism and a storage system for gathering the fuids
and waste removed from the wells. The vacuum may be
supplied by external and internal pumps. Extraction is
done by a set of needles mounted on the washer/dryers
head. The number of needles varies from one to three,
according to the washer model used.
If it uses only one needle, the washing and
extraction operation is done with this single needle.
If it uses two needles, one is used for supplying the
washing solution and the other for extraction. If it uses
three needles, the frst is used for supplying the washing
solution, the second for extraction and the third for
controlling (extracting) any excess volume in the well.
Generally, the extraction needle is longer than the
supply needle, which enables it to advance (vertically)
up to a height ranging between 0.3 and 0.5 mm from
the bottom of the well.
Advance sub-system. This is composed of a
mechanism which moves the supply and extraction
head horizontally to reach each well in the ELISA plate.
When the horizontal movement to the following row
occurs, there is a vertical movement towards the well
to dispense or extract the washing solution. There
are washers which carry out these operations in a
simultaneous manner.
The sub-systems previously described are shown in Figure
2. Figure 3 shows the diferent types of wells most commonly
found in microplates. Each kind of well is suitable for a
particular type of test.
Washing process
The washing of the microplate is one of the stages of the
ELISA technique. Special solutions are used in the washing
steps. Among those most commonly used is phosphate
bufer solution or PBS. The phosphate bufer solution has a
stability of 2 months if kept at 4 C. It is estimated that 1 to
3 litres of solution is required for washing one microplate
and that 300 l is used in each well per cycle. Washing can
be done manually, but it is preferable to use an automated
microplate washer for a better throughput and to minimize
handling of potentially contaminated substances.
Among the washing processes used by microplate washers
are featured:
Aspiration from top to bottom. When the aspiration
phase is initiated, the needles move vertically and the
aspiration is initiated immediately as these enter into
the liquid. The process continues until the needles
reach their lowest position very close to the bottom
of the wells. At this point they are stopped in order to
avoid suctioning the air that should fow against the
interior lateral walls of the wells. This type of aspiration
prevents air currents from drying the bound protein on
the surface of the wells.
Waste Receptacle
Extraction Pump
Supply and
Extraction Head
Horizontal and
Vertical
Displacement
Wells
ELISA Plate
Washing
Solution
Supply Pumps
Positive Displacement Pumps
Figure 2. Microplate washer
Flat
Bottom
Round
Bottom
V-shaped
Bottom
Easy
Wash
Figure 3. Well proles
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
9
Simultaneous distribution and aspiration. In certain
types of washer, the washing and aspiration systems
operate simultaneously, generating a controlled
turbulence inside the well which removes the unbound
substances during the incubations.
Aspiration from the base of the wells. In this system,
the aspiration of the fluid contained in the wells is
performed initially with the aspiration needles in a
position very close to the bottom, immediately
beginning a suctioning cycle, usually time-controlled.
This system may aspirate air if there are diferences in
the levels of the tanks.
Washer calibration
The microplate washer is critical for guaranteeing that the
ELISA technique performs as expected. The alignment to
be taken into account for the efective functioning of the
equipment is presented next:
Position of the needles (supply and aspiration head).
The horizontal and vertical position adjustment with
respect to the wells must be verifed carefully. If the
plate has fat bottom wells, the supply needle must be
checked to see that it is situated very close to the wells
wall. If the bottom is round or V-shaped, the suction
needle should be located in the centre of the well:
upon the vertical movement, a needle-base distance
is maintained in the well, usually between 0.3 to 0.5
mm. The needles must never be allowed to touch the
bottom of the wells to avoid mechanical interferences
between the needle point and the wells base during
the aspiration function.
Aspiration time. Appropriately adjust the aspiration
time so that a solution flm adhered to the wells wall
can fow towards the bottom. Avoid very long time
lapses to prevent the coating on the wells from drying
up. Check that the suction systems needles are clean
(free of obstructions).
Distributed Volume. Check that the volume distributed
is as close as possible to the maximum capacity of the
well; confrm that all the wells are flled uniformly (at
the same level). Verify that the distributing needles are
clean (free of obstructions).
Vacuum. The suctioning system must be calibrated
ef ciently. If the vacuum is too strong, the test can
be altered. In fact, it could dry out the wells and
considerably weaken the enzyme activity in the wells
and completely alter the test result. The majority of
washers function with a vacuum ranging between 60
and 70% of atmospheric pressure. In some washers, the
vacuum is made in an external pump which operates as
an accessory of the washer. Its operation is controlled
by the washer, which means that the vacuum pump
operates only when required.
Washing process verication
To verify that the washing process is done according to
the specifications of ELISA techniques, manufacturers
of ELISA tests have developed procedures to be carried
out regularly. One of the controls
1
is based on using the
peroxidase reagent, which is dispensed using a pipette in
the plate wells to be read at 405, 450 and 492 nm. At once
the wells are washed and a colourless substrate is added
(TMB/H2O2Tetramethylbenzidine/Hydrogen Peroxide).
Whatever conjugate remains will hydrolyze the enzyme
and the chromogen will change to blue. After stopping
the reaction with acid, the TMB will turn yellow again. The
resulting colour intensity is directly related to the washing
process ef ciency.
INSTALLATION REQUIREMENTS
For the microplate washer to operate correctly, the following
is necessary:
1. A clean, dust-free environment.
2. A stable work table located away from equipment
that generates vibrations, (centrifuges, and agitators).
It must be of a suitable size to locate the necessary
complementary equipment: reader, incubator,
distributor and computer with its peripheral attachments
at the side of the microplate washer.
3. An electric outlet in good condition with a ground pole
and, an electrical connection which complies with the
countrys or the laboratorys norms and standards. In the
countries of the Americas, the 110 V and 60 Hz frequency
is generally used. In other parts of the World, the 220-240
V and 50/60 Hz frequency is generally used.
ROUTINE MAINTENANCE
The routine maintenance described next focuses exclusively
on the microplate washer. Maintenance of the microplate
reader is dealt with in the Chapter 1.
Basic maintenance
Frequency: Daily
1. Verify the volume distributed.
2. Test the flling uniformity.
3. Verify the aspiration sub-systems ef ciency.
4. Confirm the cleaning of the supply and extraction
needles.
5. Clean the washer with distilled water after use, to remove
every vestige of salt in the supply and extraction sub-
systems channels. The needles may be kept submerged
in distilled water.
6. Verify that the body of the washer has been cleaned.
If necessary, clean the exterior surfaces with a piece of
cloth, moistened with a mild detergent.
1
Procedure developed by PANBIO, ELISA Check Plus, Cat. N E-ECP01T.
CHAPTER 2 MI CROPLATE WASHER
10
Preventive maintenance
Frequency: Quarterly
1. Disassemble and clean the channels and connectors.
Verify their integrity. If leaks or any vestiges of corrosion
are detected, adjust and/or replace.
2. Verify the integrity of the mechanical components.
Lubri cate accordi ng to the manufacturer s
instructions.
3. Test the adjustment of each one of the sub-
systems. Calibrate according to the manufacturers
recommendations.
4. Confrm the integrity of the electrical connector and the
inter-connection cable.
5. Clean the washer with distilled water after using it in
order to remove every vestige of salt in the supply and
extraction subsystems channels.
6. Verify the integrity of the fuse, and that its contact
points are clean.
Note: Trained technical personnel must carry out
maintenance of the control system. If necessary, call the
manufacturer or representative.
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
11
TROUBLESHOOTING TABLE
PROBLEM PROBABLE CAUSE SOLUTION
Upon completion of washing, residual solution
remains in the wells.
The washer extraction system demonstrates failure. Verify if the vacuum system is functioning at the
appropriate pressure.
The conducts/pipes of the vacuum system are of a
diferent diameter than that recommended.
Check that the diameter of the channels corresponds
to the recommendation by the manufacturer.
The suction line shows obstructions. Verify that the vacuum lines are clean.
The container for storing the waste is full. Confrm the waste recipients level.
The line flter is damp or blocked. Verify the state and integrity of the suctioning
systems flter.
The needles points are not placed correctly and do
not reach the bottom of the wells.
Examine the placement of the needles points.
A diferent microplate is used in the test. Verify the type of plate required for the test.
The washer has not been purged suf ciently. Check the purging process.
The operator has not followed the manufacturers
instructions correctly.
Examine the process recommended by the
manufacturer. Carry out the required adjustments.
The plate placed in the washer is incorrectly aligned. Check the placement of the plate in the washer.
The washing cycle is performing inadequately. The washing solution reserve is exhausted. Examine the cleaning solution storage receptacle.
Replace the volume missing.
The washer was not purged suf ciently at the
beginning of the work cycle.
Clean adequately in order to homogenize the
humidity in each one of its components and to
eliminate air bubbles.
The volume of washing solution distributed has been
programmed erroneously.
Verify the required volume for each type of test and
for each plate.
The plate was placed incorrectly in the washer. Check the correct installation of the plate in the
washer.
The cycle setting was incorrectly selected. Review the cycle setting recommended for each type
of plate.
The plates used are diferent from those
recommended by the manufacturer.
Verify that the plates used are completely
compatible with the washer.
The fuid level in the wells is inadequate.
The washing solution supply tube is not of the
diameter or thickness specifed by the manufacturer.
Check the manufacturers specifcations. If necessary,
correct.
The pressure is insuf cient for delivering the
adequate amount of washing solution.
Check the supply system and supply channels, there
might be an obstruction in the flling line.
The washing container shows fungal and bacterial
growths.
The system is not used frequently. Check the procedures used for preventing fungal and
bacterial growth.
An adequate control procedure (disinfection) is not
used.
Check the procedures used for preventing fungal and
bacterial growth.
The tubes and connectors are not changed with the
required frequency.
Verify the change frequency suggested by the
manufacturer and or the technical department.
The washing solution has been contaminated. Confrm the procedures used in the preparation
and management of the washing solution with the
aim of determining the cause of contamination and
eliminate it.
Maintenance has not been carried out according to
its schedule.
Check the dates planned for carrying out
maintenance. Inform those responsible.
CHAPTER 2 MI CROPLATE WASHER
12
BASIC DEFINITIONS
Bufer. A solution containing either a weak acid and its salt or, a weak base and its salt, which makes it resistant to changes in pH at a given temperature.
PBS. One of the solutions used to perform washing operations in ELISA tests. PBS is the acronym for Phosphate Bufer Solution. This is made of the following
substances: NaCl, KCl, NaHPO
4
2H
2
O and KH
2
SO
4
. The manufacturers supply technical bulletins which indicate the proportions and instructions for preparing PBS. In
general, one part of concentrated PBS is mixed with 19 parts of deionised water.
Plate (ELISA). Consumable with standard dimensions, designed to hold samples and reactions for the ELISA technique. In general, these have 96, 384 or 1536 wells
and are made of plastics such as polystyrene and polypropylene. There are plates specially treated to facilitate the performance of the tests.
Positive displacement pump. A pump adjusted by a plunger moving along a cylinder. The mechanism is similar to that of a syringe. It is equipped with a set of
valves for controlling the fow to and from the pump.
TMB/H
2
O
2
. (Tetramethylbenzidine/hydrogen peroxide). A set of reagents used for verifying the quality of washing done on the wells used in the ELISA technique.
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
13
Chapter 3
pH Meter
The pH meter is used for determining the concentration of
hydrogen ions [H
+
] in a solution. This equipment, provided
it is carefully used and calibrated, measures the acidity of
an aqueous solution. pH meters are sometimes called pH
analysers, pH monitors or potentiometers.
PURPOSE OF THE EQUIPMENT
The pH meter is commonly used in any feld of science
related to aqueous solutions. It is used in areas such as
agriculture, water treatment and purifcation, in industrial
processes such as petrochemicals, paper manufacture,
foods, pharmaceuticals, research and development, metal
mechanics, etc. In the health laboratory, its applications
are related to the control of culture mediums and to the
measurement of the alkalinity or acidity of broths and
bufers. In specialized laboratories, diagnostic equipment
microelectrodes are used to measure the pH of liquid
blood components. The plasma pH allows the patients
health to be evaluated. It normally measures between 7.35
and 7.45. This value relates to the patients metabolism
in which a multitude of reactions occurs where acids and
bases are normally kept in balance. Acids constantly liberate
hydrogen ions [H
+
] and the organism neutralizes or balances
acidity by liberating bicarbonate ions [HCO
3

]. The acid-base
ratio in the organism is maintained by the kidneys, (organs
in which any excesses present are eliminated). The plasma
pH is one of the characteristics that vary with factors such
as age or state of health of the patient. Table 1 shows typical
pH values of some bodily fuids.
PHOTOGRAPH AND COMPONENTS OF THE
pH METER
OPERATION PRINCIPLES
The pH meter measures the concentration of hydrogen ions
[H
+
] using an ion-sensitive electrode. Under ideal conditions,
this electrode should respond in the presence of only
one type of ion. In reality, there are always interactions or
interferences with other types of ions present in the solution.
A pH electrode is generally a combined electrode, in which
a reference electrode and an internal glass electrode are
integrated into a combined probe. The lower part of the
probe ends in a round bulb of thin glass where the tip
of the internal electrode is found. The body of the probe
Fluid pH Value
Bile 7.8 8.6
Saliva 6.4 6.8
Urine 5.5 7.0
Gastric Juice 1.5 1.8
Blood 7.35 7.45
pH values of some bodily uids
GMDN Code 15164
ECRI Code 15-164
Denomination pH Meter
P
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1 Electrode carrying arm and electrode
2 Digital display
3 Control panel with temperature adjustment control, mode
selection (Standby/mV/pH) and calibration controls
1
3
2
CHAPTER 3 pH METER
14
contains saturated potassium chloride (KCl) and a solution
0.1 M of hydrogen chloride (HCl). The tip of the reference
electrodes cathode is inside the body of the probe. On the
outside and end of the inner tube is the anodized end. The
reference electrode is usually made of the same type of
material as the internal electrode. Both tubes, interior and
exterior, contain a reference solution. Only the outer tube
has contact with the measured solution through a porous
cap which acts as a saline bridge.
This device acts like a galvanized cell. The reference electrode
is the internal tube of the pH meter probe, which cannot lose
ions through interactions with the surrounding environment.
Therefore as a reference, it remains static (unchangeable)
during the measuring process. The external tube of the
probe contains the medium which is allowed to mix with
the external environment. As a result, this tube must be
flled periodically with a potassium chloride solution (KCI)
for restoring the capacity of the electrode which would
otherwise be inhibited by a loss of ions and evaporation.
The glass bulb on the lower part of the pH electrode acts
as a measuring element and is covered with a layer of
hydrated gel on its exterior and interior. Metallic sodium
cations [Na
+
] are difused in the hydrated gel outside of
the glass and in the solution, while the hydrogen ions [H
+
]
are difused in the gel. This gel makes the pH electrode
ion-selective: Hydrogen ions [H
+
] cannot pass through the
glass membrane of the pH electrode. Sodium ions [Na
+
] pass
through and cause a change in free energy, which the pH
meter measures. A brief explanation of the theory on how
electrodes function is included in the appendix at the end
of the chapter.
pH METER COMPONENTS
A pH meter generally has the following components:
1. The body of the instrument containing the circuits,
controls, connectors, display screens and measuring
scales. The following are among some of its most
important components:
a) An ON and OFF switch. Not all pH meters have an
on and of switch. Some simply have a cord with a
plug which allows it to be connected to a suitable
electrical outlet.
b) Temperature control. This control allows
adjustments according to the temperature of the
solution measured.
c) Calibration controls. Depending on the design,
pH meters possess one or two calibration buttons
or dials. Normally these are identifed by Cal 1 and
Cal 2. If the pH meter is calibrated using only one
solution, the Cal 1 button is used; making sure
that Cal 2 is set at a 100%. If the pH meter allows
two point calibrations, two known pH solutions
covering the range of pH to be measured are used.
In this case, the two controls are used (Cal 1 and Cal
2). In special cases, a three-point calibration must
be done (using three known pH solutions).
d) Mode selector. The functions generally included
in this control are:
I. Standby mode (0). In this position the electrodes
are protected from electrical currents. It is the
position used for maintaining the equipment
while stored.
II. pH mode. In this position the equipment can
take pH measurements after performing the
required calibration procedures.
4
KCI KCI
High Impedance
Voltmeter
Temperature
Regulator
Reference
Terminal
Saline Mesh Bridge
Solution Under Analysis
Special Glass Permeable to Ions
Active Termimal
Ag/AgCI Electrode
Figure 4. Diagram of a pH meter
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
15
III. Millivolt mode (mV). In this position the
equipment is capable of performing millivoltage
readings.
IV. ATC mode. The automatic temperature control
mode is used when the pH is measured in
solutions for which the temperature varies. This
function requires the use of a special probe. Not
all pH meters have this control.
2. A combined electrode or probe. This device must
be stored in distilled water and stay connected to the
measuring instrument. A combination electrode has a
reference electrode (also known as Calomel electrode)
and an internal electrode, integrated into the same body.
Its design varies depending on the manufacturer.
TYPICAL CIRCUIT
Figure 6 features a typical circuit adapted to the control
system of the pH meter. Each manufacturer has its own
designs and variations.
110 VAC
1N 4002
7812
3,300
mfd
0.1
mfd
3,300
mfd
0.1
mfd
7912
10K
Variable
resistor
12V
Lamp
Entrance
Reference
10K
30K
pH
560K
mV
9,09 K
1,00 K
TL081
110 V AC/ 12 V DC
Transformer
10K
Zero
3
2
7
6
Exit
pH
mV
4
5
1
Figure 6. Example of a typical pH meter control circuit
Combined Electrode
Silver Wire (Ag)
Reference Electrode
Semi-Permeable Mesh
Buer Solution
Figure 5. Types of electrodes
Platinum Wire (Pi)
Reference Electrode (Calomel)
h
Mercury [Hg]
Mercury Chloride [Hg CI]
Potassium Chloride
Porous Stopper
CHAPTER 3 pH METER
16
System Element Description
Electric feeding and correction.

110 V/12 V AC transformer.* A device converting the voltage of the 110 V to 12 V
AC network.
1N4002 rectifer diodes. Diode controlling the type of wave and guaranteeing
that is positive.
Electrolyte condensers 3300 microfarads (fd) (2). Condensers absorbing the DC voltage to the diodes.
Tri terminal regulators (7812, 7912). A device regulating the voltage resulting from the
interaction between diodes and condensers.
0.1 microfarad (fd) (2) electrolyte condensers. Devices used to achieve stability at high frequency.
12 V D C signal light. Light indicating if the equipment is ON.
Measurement of pH and millivolts. TL081 non-inverted type dual amplifer. Millivolts circuits.
(R1) 9.09 K (ohm) resistors.
(R2) 1 K (ohm) resistors.
(R3) 560 K (ohm) resistors. pH circuits.
(R4) 10 K (ohm) variable resistors.
(R5) 30 K (ohm) resistors. Ground resistance.
The circuit gain is governed by means of the
following equation:
Gain = 1+ (R3+PxR4)/R5+ (1P) xR4.
Outlet section. Low cost DC voltmeter. Permits readings in millivolts. The voltage read is 10
times that of the cell, allowing a resolution of 0.1
millivolts.
The reading is done by using carbon/quinhydrone
electrodes.
Description of typical control circuit elements
* Diferent voltage specifcations are applicable in certain regions of the World.
INSTALLATION REQUIREMENTS
The pH meter works using electric current with the following
characteristics.
Power: Single phase Voltage: 110 V or 220-230 V Frequencies;
50-60Hz depending on the World region.
There is also portable pH meters powered with batteries.
GENERAL CALIBRATION PROCEDURE
pH analyzers must be calibrated before use to guarantee
the quality and accuracy of the readings following these
procedures:
1. One point calibration. This is carried out for normal
working conditions and for normal use. It uses one
known pH reference solution.
2. Two point calibration. This is done prior to performing
very precise measurements. It uses two known pH
reference solutions. It is also done if the instrument is
used sporadically and its maintenance is not carried out
frequently.
Description of the process
Frequency: Daily
1. Calibrate the pH meter using one known pH solution
(one point calibration).
1.1 Connect the equipment to an electrical outlet with
suitable voltage.
1.2 Adjust the temperature sel ector to the
environmental temperature.
1.3 Adjust the meter.
1.4 Remove the electrodes from the storage container.
The electrodes must always be stored in a suitable
solution. Some can be maintained in distilled
water, others must be kept in a diferent solution
as their manufacturers recommend
1
. If for some
reason, the electrode becomes dry, it is necessary
to soak it for at least 24 hours before use.
1.5 Rinse the electrode with distilled water in an empty
beaker.
1.6 Dry the electrode with material able to absorb
residual liquid on its surface, without impregnating
the electrode. To avoid possible contamination,
the electrodes must be rinsed between diferent
solutions.
1
Verify the type of bufer solution recommended by the electrode
manufacturer.
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
17
2. Place electrodes in the calibration solution.
2.1 Submerge the electrode in the standardization
solution in such a manner that its lower extremity
does not touch the bottom of the beaker. This
decreases the risk of breaking the electrode. If the
test requires that the solution be kept in motion
using the magnetic agitator, special care must be
taken so that the agitation rod does not hit the
electrode as this could break it. Bufer solution
is used as a calibration solution, because its pH
is known and therefore will still be maintained
even if a little contamination occurs. In general, a
solution of pH = 7 is used for this purpose
1
.
3. Turn the functions selector from Standby position to
pH position.
3.1 This action connects the electrode to the pH
measuring scale in the pH meter.
3.2 Adjust the meter to read the pH of the calibration
solution using the button marked Cal 1. This
enables the meter to read the pH of the calibration
solution.
For example: For a solution at pH = 7, the needle
can oscillate slightly in units of 0.1 pH; on average,
the reading should be 7. The reading of the meter
(reading scale) should be done perpendicularly,
to avoid or eliminate parallel-type errors (reading
errors produced by the shadow of the meters
needle, visible on the mirror of the reading scale).
The pH meter is then ready (calibrated), to carry
out the correct pH readings.
3.3. Put the functions selector in the Standby
position.
4. Measuring the pH of a solution.
4.1 Remove the electrode from the calibration
solution.
4.2 Rinse the electrode with distilled water and dry
it.
4.3 Place the electrode in the solution of unknown
pH.
4.4 Turn the functions selector from the Standby
position to the pH position.
4.5 Read the pH of the solution on the meters scale or
the screen. Register the reading obtained on the
control sheet.
4.6 Turn the functions selector again to the Standby
position.
If it is necessary to measure the pH of more
than one solution, repeat the previously described
procedures, rinsing the probe with distilled water
and drying with clean, lint-free paper between
readings. When the pH has to be measured
in numerous solutions, the pH meter must be
calibrated frequently, following the steps previously
described.
5. Turn off the pH meter.
5.1 Remove the electrode from the last solution
analyzed.
5.2 Rinse the electrode in distilled water and dry it with
a drying material that will not penetrate it.
5.3 Place the electrode in its storage container.
5.4 Verify that the functions selector is in the Standby
position.
5.5 Activate the off switch or disconnect the feed
cable, if it lacks this control.
5.6 Clean the work area.
GENERAL MAINTENANCE OF THE pH METER
pH meters have two general maintenance procedures:
one concerning the analyzers body, the other for the pH
detection probe (electrodes).
General maintenance procedures for the pH meters
body
Frequency: Every six months
1. Examine the exterior of the equipment and evaluate its
general physical condition. Verify the cleanliness of the
covers and their adjustments.
2. Test the connection cable and its system of connections.
Check that they are in good condition and clean.
3. Examine the equipment controls. Verify that these are
in good condition and activated without dif culty.
4. Verify that the meter is in good condition. To do this, the
instrument must be disconnected from the electric feed
line. Adjust the indicator needle to zero (0) using the
adjustment screw generally found below the pivot of
the indicator needle. If the equipment has an indicator
screen, check that it is functioning normally.
5. Confrm that the on indicator (bulb or diode) operates
normally.
6. Verify the state of the electrode carrying arm. Examine
the electrode attachment and assembly mechanism to
prevent the electrode from becoming loose. Check that
the height adjustment operates correctly.
7. Check the batteries (if applicable); change them if
necessary.
8. Test its function by measuring the pH of a known
solution.
9. Inspect the ground connection and check for escaping
current.
1
Verify the type of calibration solution recommended by the electrode
manufacturer.
CHAPTER 3 pH METER
18
BASIC MAINTENANCE OF THE ELECTRODE
Frequency: Every four months
The measuring or detector electrode requires periodic
maintenance of the conducting solution to obtain precise
readings.
The recommended steps for replacing the electrolyte
solution are the following:
1. Remove the detector electrode from the storage bufer
solution.
2. Rinse the detector electrode abundantly with distilled
water.
3. Remove the upper cover of the detector electrode.
4. Fill the conduit surrounding the internal electrode with
a saturated potassium chloride (KCI) solution. Use the
syringe or applicator supplied with the KCI solution.
Verify that the tip of the syringe does not touch the
inside of the electrode.
5. Close the electrode with its cover. Rinse the electrode
in distilled water.
6. Keep the electrode in storage bufer solution while not
in use.
Cleaning of the electrode
The type of cleaning required for electrodes depends of
the type of contaminant afecting it. The most common
procedures are summarized next:
1. General cleaning. Soak the pH electrode in a 0.1 M HCl
solution or 0.1 M HNO
3
, for 20 minutes. Rinse with
water.
2. Removal of deposits and bacteria. Soak the pH
electrode in a diluted domestic bleach solution (e.g.
1%), for 10 minutes. Rinse abundantly with water.
3. Cleaning oil and grease. Rinse the pH electrode with
a mild detergent or with methyl alcohol. Rinse with
water.
4. Cleaning of protein deposits. Soak the pH electrode
in 1% pepsin and 0.1 M HCl for 5 minutes. Rinse with
water.
After carrying out each cleaning operation, rinse with
deionised water and refll the reference electrode before
use.
Other precautionary measures
1. Do not strike the electrode. Given that the structure is
generally made of glass and very fragile, it is necessary
to manipulate it very carefully, preventing it from being
knocked of.
2. Remember that the electrode has a limited lifespan.
3. While not in use, keep the electrode inside the storage
bufer solution.
TROUBLESHOOTING TABLE
PROBLEM PROBABLE CAUSE SOLUTION
The pH meter shows unstable readings. There are air bubbles in the electrode. Soak the electrode to eliminate the bubbles.
The electrode is dirty. Clean the electrode and recalibrate.
The electrode is not immersed. Verify that the sample covers the tip of the electrode
perfectly.
The electrode is broken. Replace the electrode.
The electrodes response is slow. The electrode is dirty or greasy. Clean the electrode and recalibrate.
The screen shows an error message. Incorrect operating mode selected. Verify the operation mode selected. Select a valid
operation.
The screen shows a calibration or error message. There is a calibration error. Recalibrate the pH meter.
The calibration of the bufer value is erroneous. Verify the bufer values used.
The electrode is dirty. Clean and calibrate the electrode.
The pH meter is on, but there is no signal on the
screen.*
The batteries are badly installed. Verify the polarity of the batteries.
The batteries are worn out. Replace the batteries.
The battery indicator is fashing.* The batteries are worn out. Replace the batteries.
* Applicable to equipment equipped with batteries only.
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
19
BASIC DEFINITIONS
Bufer. A solution containing either a weak acid and its salt or, a weak base and its salt, which makes it resistant to changes in pH at a given temperature.
Calomel electrode. A reference electrode used with the active electrode for determining the pH of a solution. This electrode is constructed with a mercury base
(Hg), a covering of dimercuric chloride (Hg
2
Cl
2
) and a potassium chloride solution of 0.1 M. It is represented as Cl
2
[Hg
2
Cl
2
, KCl]Hg.
Dissociation. A phenomenon through which a break in the molecules occurs. As a result it produces electrically charged particles (ions).
Electrolyte. A solute which produces a conducting solution, e.g. NaCl (sodium chloride) and NH
4
OH.
Gel. A semisolid substance (e.g. jelly) composed of a colloid (solid) dispersed in a liquid medium.
Ion. Neutral atom which gains or loses an electron. When the atom loses an electron, it becomes a positively charged ion, called a cation. If the atom gains or captures
an electron, it becomes a negatively charged ion, called an anion.
Ion-sensitive electrode. A device which produces a diference in potential proportional to the concentration of an analyte.
Molarity. Number of Moles (M) in a substance in a litre of solution. (Number of moles of solute in a litre (L) of solution). The brackets around the ionic symbol
indicate that it is treated as a molar concentration.
Mol. (abbreviation for molecule). A quantity of any substance whose mass expressed in grams is numerically equal to its atomic mass.
Mole (unit). The amount of a substance that contains as many atoms, molecules, ions, or other elementary units as the number of atoms in 0.012 kilogram of
carbon 12. It corresponds to the number 6.0225 10
23
, or Avogadros number, also called gram molecule.
The mass in grams of this amount of a substance, numerically equal to the molecular weight of the substance, also called gram-molecular weight.
pH. Measurement of the concentration of the hydrogen ion (H
+
) given in moles per litre (M) in a solution. The pH concept was proposed by Srensen and Lindstrm-
Lang in 1909 to facilitate expressing very low ion concentrations. It is defned by the following equation:
pH = log [H
+
] or [H
+
] = 10
-pH

It measures the acidity of a solution. Example, in water the concentration of [H
+
] is 1.0 x 10
-7
M resulting in pH = 7. This allows the range of concentrations from
1 to 10
-14
M, to be expressed from zero (0) to 14. There are diverse systems for measuring the acidity of a solution. An acidic substance dissolved in water is capable
of producing H
+
ions. A basic substance dissolved in water is capable of producing [OH

] (hydroxides) ions.
An acid substance has a greater quantity of ions [H
+
] than pure water; a basic substance shows greater quantities of ions [OH

] than pure water. The concentrations


of substances are expressed in moles per litre.
In pure water, the ion concentration [H
+
] and [OH

] is 1.0 x 10
7
M, it is thus considered a neutral substance. In reality, it is a weak electrolyte that is dissociated
following the following equation:
H
2
O [H
+
][OH

]
In all aqueous solutions there is a balance expressed as:
[H
+
][OH

]
= K

H
2
O
If the solution is diluted, the concentration of the non-dissociated water can be considered constant:
[H
+
][OH

] = [H
2
O]K = K
a
The new constant Ka is called a constant of dissociation or ionic product of water and its value is 1.0x10
14
at 25 C.
[H
+
][OH

] = 1.0 x 10
-14
X x X = 1.0 x 10
-14
X
2
= 1.0 x 10
-14
X = 1.0 x 10
-7
In pure water the concentrations of H
+
and OH

are 1.0 x 10
7
M, a very low concentration, given that the molar concentration of water is 55.4 mol/litre.
Solution. Homogenous liquid mixture (with uniform properties) of two or more substances. It is characterized by the absence of chemical reactions among the
components in the mixture. The component in greater proportion and generally in a liquid state is called solvent and that or those in a lesser quantity, the solutes.
CHAPTER 3 pH METER
20
Annex
The pH theory
pH electrodes ideally behave as an electrochemical cell and react to the concentration of ions [H
+
]. This generates an
electromotive force (EMF) which, according to the Nernst law is calculated using the following equation:

Given that:
pH = lna
H
L
where a is the efective concentration of ions (Activity)
If n = 1, the equation is then rewritten as:

E = E
o

R' T
F
pH
E is a constant dependant on the temperature. If E is substituted by ET, the calibration will be more sensitive. Real electrodes
do not always perform according to the Nernst equation. If the concept of sensibility (s) is introduced, the equation can be
rewritten as:
E = E ' T s
R' T
F
pH
The values of E and s are found when measuring the EMF in two solutions with known pH. S is the slope of E versus pH,
while E is found at the intersection with the axis y. When E and s are known, the equation can be rewritten and the pH can
be calculated as:
pH =
E ' T E
s
R' T
T
E = E
o
+
RT
nF
lna
H
pH = lna
H
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
21
Chapter 4
Balances
The balance is an instrument which measures the mass of
a body or substance using the gravity force which acts on
that body. The word comes from the Latin terms bis which
means two and lanx, plate. The balance has other names
such as scale and weight. It must be taken into account that
the weight is the force which the gravitational feld exercises
on a bodys mass, this force being the product of the mass
by the local acceleration of gravity [F = m x g]. The term
local is used to emphasize that this acceleration depends
on factors such as the geographical latitude, altitude and
the Earths density where the measurement is taken. This
force is measured in Newtons.
GMDN Code 10261 10263 45513 46548
ECRI Code 10-261 10-263 18-449 18-451
Denomination Balances Electronic balances Analytical electronic
balances
Micro analytical,
microelectronic
balances
P
h
o
t
o

c
o
u
r
t
e
s
y

o
f

A
c
c
u
l
a
b

C
o
r
p
o
r
a
t
i
o
n
P
h
o
t
o

c
o
u
r
t
e
s
y

o
f

O
h
a
u
s

C
o
r
p
o
r
a
t
i
o
n
P
h
o
t
o

c
o
u
r
t
e
s
y

o
f

O
h
a
u
s

C
o
r
p
o
r
a
t
i
o
n
PHOTOGRAPHS OF BALANCES
Mechanical balance Electronic balance
CHAPTER 4 BALANCES
22
PURPOSE OF THE BALANCE
The balance is used for measuring the mass of a body or
substance or its weight. In the laboratory, the balance is used
for weighing as part of quality control activities (on devices
like pipettes), in the preparation of mixtures of components
in predefined proportions and in the determination of
specifc densities or weights.
OPERATION PRINCIPLES
There are diferences in design, principles and criteria of
metrology amongst balances. At present, there are two large
groups of balances: mechanical and electronic balances.
Mechanical balances
The following are some of the more common ones:
1. Spring balance. Its function is based on a mechanical
property of springs as the force exercised on a spring
is proportional to the springs elasticity constant [k],
multiplied by its elongation [x] [F = -kx]. The greater
the mass [m] placed on the balances plate, the greater
the elongation will be, given that the elongation is
proportional to the mass and the springs constant. The
calibration of a spring balance depends on the force
of gravity acting on the object weighed. This type of
balance is used when great precision is not necessary.
2. Sliding weight balance. This type of balance is
equipped with two known weights which can be moved
on setting scales (one macro, the other micro). Upon
placing a substance of unknown mass on the tray, its
weight is determined by moving the weight on both
setting scales until the equilibrium position is reached.
At this point, the weight is obtained by adding both
quantities indicated by the sliding masses position on
the scale.
3. Analytical balance. This balance functions by comparing
known weight masses with that of a substance of
unknown weight. It is composed of a base on a bar or
symmetrical lever, maintained by a blade-like support
on a central point called a fulcrum. At its ends, there
are stirrups, also supported with blades which allow
these to oscillate smoothly. From there, two plates
are suspended. Certifed weights are placed on one
of the plates and unknown weights on the other. The
balance has a securing system or lock, which allows
the main lever to remain stable when not in use or
when it is necessary to modify the counter-weights. The
balance is inside an external box which protects it from
interferences, such as air currents. Analytical balances
can weigh ten thousandths of a gram (0.0001 g) or 100
thousandths of a gram (0.00001 g). This type of balance
generally has a capacity of up to 200 grams.
X
Spring Without Load
Displacement
Measuring Scale
Mass
F=mg
F=-kx
Spring With Load
m
F = F1
-kx = mg
Figure 7. Spring balance
Tray
Macro Scale
Micro Sliding Weight
Macro Sliding Weight
Micro Scale
Figure 8. Sliding weight scale
Figure 9. Analytical balance
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
23
It is necessary to have a set of certifed masses. The set is
generally composed of the following pieces:
4. Upper plate balance (Top loading or parallel guidance
balance). This type of balance has a loading plate located
on its upper part, supported by a column maintained in
a vertical position by two pairs of guides with fexible
connections. The efect of the force produced by the
mass is transmitted from a point on the vertical column
directly or by some mechanical means to the loading
cell. The requirement with this type of mechanism is that
parallel guides must be maintained with exactitude of up
to 1 m. Deviations in parallelism cause an error known
as lateral load (when the mass being weighed shows
diferences if the reading is taken at the centre of the
plate or on one of its sides). The diagram shown below
explains the operation principle some manufacturers
have introduced in electronic balances.
5. Substitution Balance (Unequal-lever arm or two-
knife balance). This is a balance with a single plate.
An unknown mass is placed on the weighing plate.
It is weighed by removing known masses from the
counterweight side until it reaches a balanced position,
using a mechanical system of cams. The fulcrum is
generally of-centre in relation to the length of the load
beam and located near the front of the balance. When
a mass is placed on the weight plate and the balances
locking mechanism is released, the movement of the
load beam is projected through an optical system to a
screen located on the front part of the instrument.
Operation verication
The procedure used for verifying the functioning of a typical
mechanical balance is described below. The described
process is based on the substitution balance.
1. Verify that the balance is levelled. The levelling is
achieved using a ring-shaped adjustment mechanism
located on the base of the balance or by adjusting a
bubble or knob on a scale located on the front of the
balances base.
2. Test the zero mechanism. Place the controls on zero
and free the balance. If the reading does not stay at zero,
adjust the zero mechanism (a grooved screw located in
a horizontal position near the fulcrum). To do this, it is
necessary to block the balance and slightly adjust the
mechanism. The process is to be continued until the
zero adjusts correctly on the reading scale.
3. Verify and adjust the sensitivity. This is always readjusted
whenever some internal adjustment is done. It is
performed with a known standard according to the
following steps:
a) Lock the balance.
b) Place a standard weight (equivalent to the optical
scale range) on the plate.
c) Position the micro setting to one (1).
d) Release the balance.
e) Adjust to the zero position.
f ) Position the micro setting to zero (0). The balance
should indicate 100. If the scale displays less
or more than 100, the sensitivity control must
be adjusted. This requires locking the balance,
opening the upper cover and turning the sensitivity
screw: If the scale registers more than 100; turn
the screw in a clockwise position. If the scale
registers less than 100, it is necessary to unwind
the screw anticlockwise. Repeat the process until
the balance is adjusted (adjusting the zero and the
sensitivity).
Type of mass Capacity
Simple pieces

1, 2, 5, 10, 20, and 50 g
100, 200 and 500 g
Fractional pieces

2, 5, 10, 20 and 50 mg
100, 200 and 500 mg
Mass
Plate
Flexible
Connections
Support Column
F
G
Figure 10. Upper plate balance
Figure 11. Substitution balance
CHAPTER 4 BALANCES
24
4. Verify the plates brake. It is mounted on a threaded
axis which touches the plate in order to prevent it from
oscillating when the balance is locked. In case of an
imbalance, the axis must be rotated slightly until the
distance between the break and the plate is zero when
the balance is locked.
Maintenance of the mechanical balance
The maintenance of mechanical balances is limited to the
following routines:
Frequency: Daily
1. Verify the level.
2. Verify the zero setting.
3. Verify the sensitivity adjustment.
4. Clean the weighing plate.
Frequency: Annually
1. Calibrate the balance and document the process.
2. Disassemble and clean the internal components. This
must be done according to the process outlined by the
manufacturer or a specialized frm must be contracted
to do so.
Electronic balances
The electronic balances have three basic components:
1. A weighing plate. The object to be weighed placed
on the weighing plate exercises a pressure distributed
randomly over the surface of the plate. By means of
a transfer mechanism (levers, supports, guides), the
weights load is concentrated on a simple force [F] which
can be measured. [F = Pa]. The pressures integral part
on the area allows the force to be calculated.
2. A measuring device known as load cell produces an
exit signal corresponding to the loads force in the form
of changes in the voltage or frequency.
3. A digital analogous electronic circuit shows the fnal
result of the weight digitally.
Laboratory balances operate according to the principle
of compensation of the electromagnetic force applicable
to displacements or torques. The combination of their
mechanical components and automatic reading systems
provides weight measurements at defned levels of accuracy
depending on the model.
Principle. The mobile parts (weighing plate, support
column [a], bobbin, position and load indicator [G] -the
object in the process of being weighed-) are maintained
in equilibrium by a compensation force [F] equal to the
weight. The compensation force is generated by an electrical
current through a bobbin in the air gap of a cylindrical
electromagnet. The force F is calculated with the equation
[F = I x L x B] where: I = electrical intensity, L = total length
of the wire of the coil and B = magnetic fow intensity in the
electromagnets air gap.
With any change in the load (weight/mass), the mobile
mechanical system responds by moving vertically a fraction
of distance. Detected by a photosensor [e], an electrical
signal is sent to the servo-amplifer [f ]. This changes the
fow of electrical current passing through the bobbin of the
magnet [c] in such a manner that the mobile system returns
to the balanced position upon adjusting of the magnetic
fow in the electromagnet. Consequently, the weight of
the mass [G] can be measured indirectly at the start of the
electrical current fow, which passes through the circuit
measuring the voltage [V] by means of a precision resistor
[R], [V = I x R]. To date, many systems developed use the
electronic system for carrying out very exact measurements
of mass and weight. The following diagram explains how
electronic balances function.
Transfer
Mechanism
Load Cell
Screen and
Signal Processor
P
Figure 12. Components of electronic balances
G
b
a
e
f
c
d
R V=I*R
I
Figure 13. Compensation force principle
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
25
The signal processing system
The signal processing system is composed of the circuit which
transforms the electrical signal emitted by the transducer
into numerical data which can be read on a screen. The
signal process comprises the following functions:
1. Tare setting. This setting is used to adjust the reading
value at zero with any load within the balances capacity
range. It is controlled by a button generally located on
the front part of the balance. It is commonly used for
taring the weighing container.
2. Repeatability setting control. During a reading, weighed
values are averaged within a predefned period of time.
This function is very useful when weighing operations
need to be carried out in unstable conditions, e.g. in
the presence of air currents or vibrations. This control
defnes the time period allowed for a result to lie within
preset limits for it to be considered stable. In addition,
it can be adjusted to suit a particular application.
3. Rounding off. In general, electronic balances process
data internally at a greater resolution than shown on the
screen. The internal net value rounded of is displayed
on the screen.
4. Stability detector. This light indicator fades when the
weighing result becomes stable and is ready to be
read. Alternatively in other balance models, this feature
allows the display of the result on the screen when the
measure of the weight becomes stable.
5. Electronic signalling process. It allows the processing
and display of the weighing operation results. It may also
allow other special functions such as piece counting,
percentage weighing, dynamic weighing of unstable
weight (e.g. animals), and formula weighing, among
others. The calculations are done by the microprocessor
following the instructions entered by the operator on
the balances keyboard.
Classication of balances
The International Organization of Legal Metrology (OIML)
has classifed the balances into four groups:
Group I: special exactitude
Group II: high exactitude
Group III: medium exactitude
Group IV: ordinary exactitude
The graph in Figure 14 shows the above-mentioned
classifcation.
In the metrological classifcation of electronic balances, only
two parameters are of importance:
1. The maximum load [Max.]
2. The value of the digital division [d]
1
The number of the scales divisions is calculated by means
of the following formula.
n =
Max
d
d
The OIML accepts the following convention for laboratory
balances.
1. Ultramicroanalytics d
d
= 0.1 g
2. Microanalytics d
d
= 1 g
3. Semi-microanalytics d
d
= 0.01 mg
4. Macroanalytics d
d
= 0.1 mg
5. Precision d
d
1 mg
1
Kupper, W., Balances and Weighing, Mettler Instrument Corp., Princeton-
Hightstown, NJ.
Figure 14. Classication of balances by exactitude
CHAPTER 4 BALANCES
26
Electronic balance controls
A diagram of the typical controls on a modern electronic
balance is shown in Figure 15. From this diagram it is
necessary to point out the following:
1. Numerous functions are incorporated.
2. Various measuring units can be selected.
3. It is possible to know the day and hour when the
measurements were taken.
4. The processes done can be documented and printed.
5. It is possible to select the language.
INSTALLATION REQUIREMENTS
For the satisfactory installation and use of a balance, the
following is required:
1. An environment with no air currents or sudden changes
in temperature and free from dust.
2. A perfectly levelled table/counter. A platform of high
inertia, isolated from the structures located in its vicinity
is ideal to reduce the efect of vibrations from certain
equipment such as centrifuges and refrigerators.
There must be a large enough area for installing the
balance and any auxiliary equipment needed during
the weighing processes. Likewise, the space required
for cables such as the interconnection, electrical current
cables and the information system connection to the
printer must be anticipated.
3. Avoid installing equipment which produces elevated
magnetic felds or vibrations like centrifuges, electrical
motors, compressors and generators in its vicinity.
4. Avoid locating it directly under the air-conditioning
system (air currents) and sunlight.
5. An electrical outlet which complies with the current
electrical standards in the country or the laboratory. It
must be in good condition and equipped with a ground
pole and switches.
Electronic balance operation
The operation of a modern electronic balance is clearly
detailed in its operators manual from the manufacturer. In
general, it must conform to the following procedure:
1. Allow the balance to equilibrate with the environment
where it is installed.
2. Allow the balance to warm-up before initiating activities.
Normally it is suf cient to have it connected to the
electrical feed system. Some manufacturers suggest at
least 20 minutes from the moment it is energized until
use. Analytical balances Class 1 require at least 2 hours
for warming before initiating use.
Verify that the balance is calibrated. Electronic
balances generally have a factory-made calibration
stored in memory which can be used if it does not
have calibration masses. If calibration is required, use
calibrated masses as indicated by the manufacturer. The
calibrated masses must conform or exceed the ASTM
tolerances. For general information, the following table
shows the accepted tolerance for the ASTM Class 1
1

masses.
3. Follow the instructions indicated in the manufacturers
operations manual.
Calibration of balances
The calibration of balances must
be done by personnel specially
trained for this activity. It should be
highlighted that it must be done
based on the alignments of the OIML
or an equivalent body such as the
American Society for Testing and
Materials (ASTM), institutions which
have developed methodologies for
classifying standard weights. The
reference weights classifcation used
by the OIML is covered in the table
opposite.
Weight (grams) Higher limit (g) Lower limit (g)
100 100.0003 99.9998
200 200.0005 199.9995
300 300.0008 299.9993
500 500.0013 499.9988
1 000 1000.0025 999.9975
2 000 2000.0050 1999.9950
3 000 3000.0075 2999.9925
5 000 5000.0125 4999.9875
1
Field Services Handbook for High Precision
Scales, IES Corporation, Portland, Oregon, 2004.
Selector
Buttons
Menu
Tare Button
Screen
Level
Selection/
Mode Button
Printing Button
Menu Button
On/O
Unit
Date Hour
Calibration
Figure 15. Analytical balance control panel
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
27
Class Description Tolerance
Uncertainty
allowed
Frequency of
recalibration
E1

Stainless steel weights without marks or adjusting
cavity.
0.5 ppm per kg 1/3 of the tolerance 2 years
E2 Stainless steel weights without marks or adjusting
cavity.
1.5 ppm per kg 1/3 of the tolerance 2 years
F1 Stainless steel weights with screw button for protecting
the adjusting cavity.
5 ppm per kg 1/5 of the tolerance 1 year
F2 Bronze plated weights. 15 ppm per kg 1/5 of the tolerance 1 year
M1 Bronze weights (that do not corrode or become stained)
or of cast iron weights with a high quality paint fnish.
50 ppm per kg 1/5 of the tolerance 1 year
M2 Bronze or cast iron weights (commercial weights). 200 ppm per 1 kg 1/5 of the tolerance 1 year
Table of OIML reference weights classication
1

Any calibration process must be done using standard
weights. The results obtained must be analyzed to determine
if these are within the acceptable tolerances. The standard
weights must be selected based on the balances capacity.
The above table complements the previous. It provides
guidance in determining the standard weights to use in the
calibration of a balance according to its capacity.
ROUTINE MAINTENANCE
The balance is characterized as an instrument of high
precision. For this reason, the operator is only responsible
for minimal maintenance limited to the following:
Daily Activities
1. Clean the weighing plate so that it is kept free of dust.
Cleaning is done by using a piece of clean cloth which
may be dampened with distilled water. If there is a stain,
a mild detergent can be applied. Also a paintbrush with
soft bristles can be used to remove particles or dust
deposited on the weight plate.
2. Clean the weighing chamber, externally and internally.
Verify that the glass is free from dust.
3. Verify that the adjustment mechanisms on the front
door of the weighing chamber works adequately.
4. Always use a clean, pre-weighed container for weighing
(glass container or weighing paper if possible). Note
that plastic can become electromagnetically charged
and is not recommended for weighing powdered or
granulated chemicals.
5. Any spill must be cleaned immediately to avoid corrosion
or contamination. Use 70% ethanol to disinfect the pan
of the balance.
Very important: Never lubricate a balance unless the
manufacturer has expressly indicated it. Any substance
interfering with the mechanism of the balance retards its
response or defnitely alters the measurement process.
Note: In general, the manufacturer or the specialized
installation representative carries out the maintenance
of the balances, according to procedures which vary
depending on the type and model.
1
Guidelines for calibration in laboratories, Drinking Water Inspectorate by
LGC (Teddington) Ltd., December 2000.
Capacity
Resolution
100 g 10 g 1 g 100 mg 10 mg 1 mg 0.1 mg 0.01 mg
Up to 200 g M1 M1 F2 F1 F2
200 g to 1 kg M1 M1 F2 F1/E2 E2 E2
1 to 30 kg M2 M2 M1 F2 E2 E2 E2
30 to 100 kg M2 M1 F2 F1 E2
More than
100 kg
M2 M1/F2 F1 E2
Table of standard weights use according to the balances capacity
CHAPTER 4 BALANCES
28
FUNCTIONAL ERROR PROBABLE CAUSE
Readings not reproducible (hysteresis). The measurement cell is dirty.
The measurement cell is badly assembled.
Non-linear readings. Defective electronic system.
Mechanical system is in bad condition.
Digital reading continually goes up or down. Defective electronic system.
Change in room temperature.
The digital reading goes up and down continually. Dirty measuring cell.
Defective electronic system.
Environmental problems like air currents, static
electricity or vibrations.
The digital screen is blank or shows marks that make
no sense.
Defective electronic system.
The screen indicates an overload or negative
condition without a load being applied.
Measuring cell damaged by overload.
Measuring cell is inadequately assembled.
The balance cannot be calibrated. Defective calibration battery.
Electronic system is defective.
Measurement cell is inadequately assembled.
TROUBLESHOOTING TABLE
Electronic balance
PROBLEM PROBABLE CAUSE SOLUTION
The balance does not turn on. T he interconnection cable is disconnected or
maladjusted on the balance.
Check the connection. Adjust the cable connector if
this is the case.
Electrical outlet has no power. Check electrical feed.
The weight reading is incorrect. The balance was not adjusted to zero before the
reading.
Place the balance on zero; repeat the measurement.
The balance is incorrectly calibrated. C alibrate according to the procedure recommended
by the manufacturer.
The balance is not levelled. Level the balance.
The balance does not show the desired units of
measurement on the screen.
The units are incorrectly selected.
select the required measurement unit.
The unit required not available or not activated. Activate the measurement unit according to the
The menu may be locked. Check to see if the locking switch is activated. If this
is the case, deactivate it.
The balance is incapable of keeping the selections
or changes. process.
Verify that the changes and selections are done
according to the manufacturers instructions. Repeat
the selection or change.
The balances reader is unstable. There is vibration on the surface of the table/counter.
again.
The front door of the balance is open.
Place the balance on a stable surface.
Close the front door to measure.
The RS232 interface does not function. The interconnection cable is maladjusted. Check the connection of the interconnection cable.
The screen shows incomplete readings or is locked. The microprocessor is locked.
the situation persists, seek technical assistance from
the service representative.
The screen displays an error code. Various. Verify the error codes in the balances manual.
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BASIC DEFINITIONS
ASTM. American Society of Testing and Materials.
Calibration. Determination of the correct value of an instruments reading by measurement or comparison against a standard or norm. A balance is calibrated by
using standard weights.
Certifed masses. Masses conforming to the tolerance defned by the certifcation bodies. The ASTM classes 1 to 4 standards are those most widely used and must
be used (a compulsory reference) for performing the calibration routines.
Exactitude. The sum of all the balances errors. This is called total error band.
Hysteresis. The diference in the results when the load in the balance is increased or decreased.
Lateral load. A balances ability to consistently read the value of masses, no matter where they are placed on the weighing scale. This is also called corner load.
Lateral load error. A deviation in the results when an object is weighed placing it in diferent parts of the weighing plate, i.e. in the centre of the plate and on
one of its sides.
Linear error. A diference showed when the balance is loaded in a successive manner, increasing the quantity of weight in equal magnitude until it reaches its
maximum capacity and unloaded in an analogous process. The diferences shown between the readings obtained and the arithmetic values corresponding to the
weights used are interpreted as non-linearity.
Linearity. Refers to the ability of a balance to perform accurate readings of weights throughout its weighing capacity . A graph showing weight compared to the
weight indication on a perfectly linear balance should generate a straight line. In order to determine the linear error of a balance, certifed masses must be used.
The procedure allows the linear diferences to be calculated by reading certifed masses with and without preloading. The diference between the readings allows
the linear error to be calculated.
Mass. A physical property of the bodies related to the quantity of matter, expressed in kilograms (kg), these contain. In physics, there are two quantities to which
the name mass is given: gravitational mass which is a measure of the way a body interacts with the gravitational feld (if the bodys mass is small, the body
experiences a weaker force than if its mass were greater) and the inertial mass, which is a quantitative or numerical measure of a bodys inertia, that is, of its
resistance to acceleration. The unit for expressing mass is the kilogram [kg].
OIML. International Of ce of Legal Metrology.
Sensitivity. The smallest mass detected by the balance or the smallest mass that the balance can measure correctly.
Sensitivity error. Constant deviation throughout the weighing range or capacity of a balance.
Traceability. The ability to relate the measurements of an instrument to a defned standard.
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Chapter 5
Water Bath
The water bath is an instrument used in the laboratory for
carrying out serological, agglutination, inactivation, bio-
medical, and pharmaceutical tests and even for industrial
incubation procedures. In general they use water, but some
baths use oil. The temperature range at which water baths
are normally used range between room temperature and
60 C. Temperatures of 100 C can be selected, using a cover
with special characteristics. Water baths are manufactured
with chambers of a capacity ranging from 2 to 30 litres.
DIAGRAM OF A WATER BATH
Below is a basic diagram of a water bath. In the
diagram, it is possible to observe the electronic
control, the screen, the cover (an optional
accessory) and the tank. Other components
can be installed, e.g. a thermometer and
an agitation unit to keep the temperature
constant (not shown).
OPERATION PRINCIPLES
Water baths are made of steel and are
generally covered with electrostatic paint
with high adherence and resistance to
environmental laboratory conditions. Water
baths have an external panel on which the
controls can be found. They also have a tank
made of rustproof material with a collection
of electrical resistors mounted on their lower
part. By means of these, heat is transferred to
the medium (water or oil) until reaching the
temperature selected with a control device
(thermostat or similar). The resistors may be
of the following types:
Immersion type. These resistors are installed inside
a sealed tube and located on the lower part of the
container in direct contact with heating medium.
External. These resistors are located on the lower part
but on the outside of the tank. These are protected by
an isolating material which prevents heat loss. This type
of resistor transfers the heat to the bottom of the tank
through thermal conduction.
The water bath is an instrument used in the laboratory for Immersion type. These resistors are installed i
GMDN Code 36754 16772
ECRI Code 15-108 16-772
Denomination Water bath Water bath, shaker
Figure 16. Water bath
31
External
Resistors
Immersion
Resistors
Figure 17. Immersion and external resistors
CHAPTER 5 WATER BATHS
32
Certain types of water bath have a series of accessories such
as agitation systems or circulators, generating carefully
controlled movement of the heating medium to keep the
temperature uniform. A table which describes the main
types of water baths is shown below.
WATER BATH CONTROLS
Water baths generally have very simple controls.
Some manufacturers have incorporated controls with
microprocessors. They vary depending on the type of
bath. The diagram of a basic water baths control panel is
shown next.
The control panel has these elements:
1. The on and of control switch
2. A Menu button for selecting the operations parameters:
operation temperature, alarm temperature, temperature
scale (C, F)
3. Two buttons for parameter adjustment
4. A screen
5. A pilot light
6. Pilots (2) for identifying the temperature scale (C, F).
WATER BATH OPERATION
Installation
1. Install the water bath close to an electrical outlet. The
outlet must have its respective ground pole in order
to guarantee the protection and safety of the operator
and the equipment. Water baths generally operate at
120 V/60 Hz or 230 V/60Hz. Its installation and use is
facilitated by a sink close by for supplying and draining
of water.
2. Verify that the location selected is levelled and has the
necessary resistance to safely support the weight of the
water bath when it is full of liquid.
3. Ensure that the location has a suitable amount of space
for putting the samples and the accessories required for
the normal operation of the water bath.
4. Avoid placing the water bath where there are strong air
currents which can interfere with its normal operation.
For example: in front of an air-conditioning unit or
window.
Safety
1. Avoid the use of the water bath in environments where
there are fammable and combustible materials. The
equipment has components (resistors generating very
high temperatures) which could start an accidental fre
or explosion.
2. Always connect the equipment to an electrical outlet
with a ground pole to protect the user and the
equipment from electrical discharges. The electrical
connection must comply with the required norms of
the country and the laboratory.
3. Use the water bath exclusively with non-corrosive or
non-fammable liquids.
4. Use personal protective elements when working with
the water bath. The bath has resistors which can cause
burns if inadvertently touched, even a considerable
time after turning of the equipment.
5. When working with substances that generate vapours,
place the water bath under a chemical hood or in a well
ventilated area.
6. Remember that liquids incubated in the water bath tank
can produce burns if hands are inadvertently placed
inside it.
7. Take into account that the water bath is designed for
use with a liquid inside the tank. If the inside is dry, the
temperature of the tank can become very high. Use
the difusing tray for placing the container inside of the
flled tank of the water bath. This has been designed for
distributing the temperature in a uniform way.
8. Avoid using the water bath if any of its controls is not
working, e.g. the temperature or limit controls.
Class Temperature range
Low temperature Room temperature up to 60 C
Room temperature up to 100 C
High temperature Room temperature up to 275 C. When it needs to
reach temperatures above 100 C, it is necessary to use
fuids other than water as the boiling point of water is
100 C under normal conditions
This type of bath generally uses oils which have much
higher boiling points.
Insulated Room temperature up to 100 C with accessories and/
or agitation systems (with water).
4. Screen 1. On and O Switch
5. On Pilot
2. Menu Button
6. Temperature
Scale Pilots (
o
C/
o
F)
3. Parameter
Adjustment Buttons
Figure 18. Water bath controls
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Using the water bath
Before using the water bath, verify that it is clean and
that accessories needed are installed. The steps normally
followed are:
1. Fill the water bath with fuid to keep the temperature
constant (water or oil). Verify that once the containers
to be heated are placed, the fuid level is between 4 and
5 cm from the top of the tank.
2. Install the control instruments needed, such as
thermometers and circulators. Use additional mounts
provided for this purpose. Verify the position of the
thermometers bulb or thermal probe to ensure that
the readings are correct.
3. If water is used as the warming fuid, verify that it is clean.
Some manufacturers recommend adding products
which prevent the formation of fungus or algae.
4. Put the main switch N 1 in the ON position (the
numbers identifying the controls herein correspond
to those shown in the diagram). Some manufacturers
have incorporated controls with microprocessors which
initiate auto-verifcation routines once the ON switch is
activated.
5. Select the operation temperature using the Menu N 2
button and the buttons for adjusting the parameters.
6. Select the cut-of temperature (in water baths with this
control). This is a safety control which cuts of the supply
of electricity if it exceeds the selected temperature.
This is selected also by using the menu button and is
controlled by the parameter adjustment buttons.
7. Avoid using the water bath with the substances
indicated below:
a) Bleach.
b) Liquids with high chlorine content.
c) Weak saline solutions such as sodium chloride,
calcium chloride or chromium compounds.
d) Strong concentrations of any acid.
e) Strong concentrations of any salt.
f ) Weak concentrations of hydrochloric, hydrobromic,
hydroiodic, sulphuric or chromic acids.
g) Deionised water, as it causes corrosion and
perforation in the stainless steel.
Maintenance
Warning: Before carrying out any maintenance activity,
disconnect the equipment from the electrical feed outlet.
Water baths are equipment whose maintenance is simple.
The recommended routines mainly focus on the cleaning
of external components. The most common routines are
featured next.
Cleaning
Frequency: Monthly
1. Turn of and disconnect the equipment. Wait until it
cools to avoid the risk of burns and accidents.
2. Remove the fuid used for heating. If it is water, it can
be poured through a siphon. If it is oil; collect into a
container with an adequate capacity.
3. Remove the thermal difusion grid located at the bottom
of the tank.
4. Disassemble the circulator and clean to remove scale
and potential algae present.
5. Clean the interior of the tank with a mild detergent.
If there is any indication of corrosion, use substances
for cleaning stainless steel. Rub lightly with synthetic
sponges or equivalent. Avoid using steel wool to remove
rust stains as these leave particles of steel which could
accelerate corrosion.
6. Avoid bending or striking the temperature control
capillary tube generally located at the bottom of the
tank.
7. Clean the exterior and interior of the water bath with
clean water.
Lubrication
Frequency: Daily
For water baths with an agitation unit or circulator
system:
Lubricate the axis of the circulators electric motor. Put a
drop of mineral oil on the axis so that a good lubricating
condition is maintained between the motors bearings
and its axis.
Periodic inspection
Frequency: Quarterly
Check the thermometer or temperature controls every three
months using known standards. If no reference standard is
available, use an ice/water mixture and/or boiling water.
Note that the thermometer or the water bath temperature
controls should also be checked when the equipment is frst
installed after purchase.
CHAPTER 5 WATER BATHS
34
TROUBLESHOOTING TABLE
PROBLEM PROBABLE CAUSE SOLUTION
There is no power to the instrument. The water bath is disconnected. Connect the water bath.
The switch is defective. Change the switch.
The fuse is defective. Substitute the fuse.
The water bath is not getting hot. The temperature control not set. Set the temperature control.
The resistor(s) is/are defective. Change resistor(s).
The limit control is not set Set the limit control.
The temperature is higher than that selected. The temperature control is defective. Change the temperature control if required.
Verify the selection of the parameters.
The samples are warmed slowly. The tank is empty or contains very little fuid. Fill the tank up to the recommended level.
The temperature is increasing very slowly. The resistor(s) is/are defective. Change the resistor(s).
The temperature control is defective. Substitute temperature control.
BASIC DEFINITIONS
Circulator. An apparatus that shakes or stirs fuids to keep their properties (temperature, color, density) homogenous. These are also called agitators.
Difusing tray. Device located at the bottom of the water bath to support the containers located inside the tank. It also allows thermal convection currents generated
in the fuid contained in the tank to circulate from top to bottom and back to the top, maintaining the temperature homogeneous at the level selected by the operator.
In general the difusing tray is made of stainless steel.
Electrostatic painting. A painting process that uses the particle-attracting property of electrostatic charges. A potential diference of 80-150kV is applied to a
grid of wires through which the paint is sprayed to charge each particle. The metal objects to be sprayed are connected to the opposite terminal of the high-voltage
circuit, so that they attract the particles of paint. The piece covered with paint particles is then placed in an electrical oven to melt the particles, making them adhere
strongly to the piece.
Fuse. A safety device which protects the electrical circuits from excessive current. Fuses are made of materials whose dimensions and properties equip them to
work well within some predefned conditions. If for some reason the design parameters are exceeded, the material burns out and interrupts the passage of the
electrical current.
Immersion resistor. An electrical resistor (see defnition below) inside of a sealed tube. These are generally used for heating fuids as water or oil.
Resistance. Opposition that a material or electrical circuit imposes to the fow of electric current. It is the property of a circuit that transforms electrical energy
into heat as it opposes the fow of current. The resistance [R], of a body of uniform section such as a wire, is directly proportional to the length [l] and inversely
proportional to the sectional area [a]. The resistance is calculated by the following equation:
R = k
l
a
Where:
k = constant that depends on the units employed
l = Length of the conductor
a = sectional area of the conductor
The ohm () is the common unit of electrical resistance; one ohm is equal to one volt per ampere.
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Chapter 6
Biological Safety Cabinet
This equipment is designed for controlling aerosols and
microparticles associated with managing potentially
toxic or infectious biological material in laboratories in
activities such as agitation, centrifugation, pipetting, and
opening of pressurized containers. Safety cabinets have
been designed to protect the user, the environment and
the sample manipulated using appropriate ventilation
conditions. They are also known as laminar fow cabinets
and/or biosafety cabinets.
ILLUSTRATION OF A BIOLOGICAL SAFETY CABINET
PURPOSES OF THE EQUIPMENT
The biological safety cabinet is used for the following:
1. To protect the worker from risks associated with the
management of potentially infectious biological
material.
2. To protect the sample being analyzed from becoming
contaminated.
3. To protect the environment.
The cabinets are used for routine work related to pathogens
(parasites, bacteria, virus, fungus), cell culture and under
very precise conditions, the management of toxic agents.
OPERATION PRINCIPLES
The biological safety cabinet is a chamber generally
constructed of steel. It has a front glass window of adjustable
height, a ventilation system with an electrical motor, a
ventilator and a set of ducts which while functioning,
generate a negative pressure condition inside the cabinet.
This forces the air to fow from inside the cabinet through
the front opening to generate a curtain of air protecting
the operator. Internally, the air is conducted through a
series of grids and ducts to be finally treated in HEPA
1

flters. Depending on the design of the cabinet, the air is
recycled inside the laboratory or extracted and renewed in
diverse proportions. The air fow, which in Class II cabinets
moves from the flter towards the work surface, is laminar. A
summary of the existing type of cabinets and their principal
characteristics is presented next.
1
HEPA: High Ef ciency Particulate Air.
GMDN Code 15698 20652 20653 20654
ECRI Code 15-698 20-652 20-653 20-654
Denomination Cabinets, biological
safety
Cabinets, biological
safety, class I
Cabinets, biological
safety, class II
Cabinets, biological
safety, class III
Figure 19. Biological safety cabinet
CHAPTER 6 BI OLOGI CAL SAFET Y CABI NET
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Type of cabinet, with illustration Characteristics
CLASS I TYPE A
1. Protection provided: to the operator and the
environment.
2. Air velocity on entering the cabinet: 38 cm/s.
3. Suitable for working with bio-safety level
1
1, 2 or 3
agents.
4. Filtration HEPA, located in extraction system which
may or may not be connected to the exterior.
5. Disadvantage: Does not protect the sample
manipulated in the cabinet.
CLASS II TYPE A
1. Protection ofered: To the operator, the product and
environment.
2. Air velocity on entering the cabinet: 38 cm/s.
3. Suitable for working with agents with biosafety level
1, 2 or 3.
4. Filtration system: two HEPA flters, one located on the
work surface; the second on the extraction system
which may or may not be connected to the exterior.
If they are connected to the exterior, it utilizes a bell
type connection.
5. They recycle approximately 70 % of the air volume and
renew 30 % of it.
Summary of biological safety cabinet types
1
See biosafety classifcations levels of agents in the following section Biological safety.
HEPA Extraction Filter
HEPA Extraction Filter
Vertical Laminar Flow
Rear Plenum
Work Area
Rear Grid
Ventilator Motors
Ventilator Suction Mouth
HEPA Filtered Air
Front Window
Front Aperature
Air Entry
Front Grid
Potentially Contained Air
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Type of cabinet, with illustration Characteristics
CLASS II TYPE B1
1. Protection provided: to the operator, the product and
the environment.
2. Air velocity entering the cabinet: 50.8 cm/s.
3. Suitable for working with agents with biosafety level
1, 2 or 3.
4. Filtration system: Two HEPA flters. It extracts
potentially contaminated air (70 %) through a duct
and recycles inside of the cabinet, after fltering, air
taken from the exterior, through the front grid (30 %).
5. All biologically contaminated ducts have a negative
pressure.

6. Allows work with small quantities of toxic and
radioactive chemicals.
CLASS II TYPE B2
1. Protection provided: to the operator, the product and
the environment.
2. Air velocity on entering the cabinet 50.8 cm/s.
3. Suitable for working with agents of biosafety level 1, 2
or 3.

4. Filtration system: Two HEPA flters. It is known as the
total extraction cabinet. It does not have any type of
recirculation.

5. All biologically contaminated ducts have a negative
pressure.
6. It has an extraction duct which allows work with toxic
and radioactive chemicals.
Plenum System
Exyraction Duct
HEPA Filters
Laminar Flow
Work Surface
V=100 PLm
[50.8cm/s]
Prelter
Extraction Duct
HEPA
Extraction Filter
Posterior Duct with
Negative Pressure
Back Grid
Front Grid
Lateral View
HEPA
Supply Filter
V vert = 55 PLm - (28cm/s)
V = 100 PLm - (50.8cm/s)
CHAPTER 6 BI OLOGI CAL SAFET Y CABI NET
38
Type of cabinet, with illustration Characteristics
CLASS II TYPE B3 OR A/B3
1. Protection provided: to the operator, the product and
the environment.

2. Air velocity on entering the cabinet: 50.8 cm/s.

3. Suitable for working with agents of biosafety level 1, 2
or 3.
4. Filtration system: Two HEPA flters.
5. All biologically contaminated ducts have a negative
pressure.
6. It is known as a combined cabin. It can be connected
by means of a duct. It is denominated as Type B3. If the
duct is missing, it is a Type A. It recycles 70 % of the air
volume inside the cabinet.
CLASS III
1. Protection provided: to the operator, the product and
the environment.
2. Filtration system: two HEPA flters in series in the
extraction; a HEPA flter in the admission.
3. Suitable for working with agents classifed biosafety
level 4.
4. Totally sealed cabinet. The intake and extraction
elements are conducted through a double -door pass-
through box. The manipulation of materials is done by
using sealed gloves at the front of the cabinet.
HEPA Extraction Filter
HEPA Supply Filter
V vert = 55 PLm (28cm/s)
V = 100 PLm - (50.8cm/s)
Front Grid
Rear Duct with Pressure [-]
Rear Grid
LATERAL VIEW
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BIOLOGICAL SAFETY
1
Microorganisms have been classifed into four categories
based on factors such as pathogenicity, infectious doses,
transmission modes, and host range, availability of
preventive measures and efectiveness of treatment for
the disease caused.
1. Risk level 1 group is composed of biological agents
very unlikely to cause sickness in healthy humans or
animals. (No individual and community risk).
2. Risk level 2 group is composed of pathogens which
cause sickness in humans or animals but unlikely to
be dangerous to laboratory workers, the community,
domestic animals or the environment under normal
circumstances. Those exposed in the laboratory rarely
become seriously ill. There are preventive measures
and effective treatment available and the risk of
dissemination is limited. (Moderate individual risk,
limited community risk).
3. Risk level 3 group is composed of pathogens which
usually cause serious sicknesses to human beings and
animals and produce a serious economic impact.
However, infection by casual contact by one
individual to another is not common. The sicknesses
these produce are treatable by antimicrobial or anti-
parasitic agents. (High individual risk, low community
risk).
4. Risk level 4 group is composed of pathogens which
usually produce very serious sicknesses in human beings
or animals, frequently without treatments available.
These agents are easily spread from one individual
to another or from animal to human being or vice
versa, directly or indirectly or by casual contact. (High
individual risk, high community risk).
INSTALLATION REQUIREMENTS
The following are requirements for a cabinet to function
adequately:
1. A laboratory area protected from air currents from
windows or air-conditioning systems. The cabinet must
also be located far from the laboratory circulation zones
in order to avoid air currents that could affect the
curtain of air inside the cabinet. It must also be verifed
that the cabinet is not installed alongside other types
of cabinets such as chemical hoods.
2. An electrical connection equipped with the respective
control and safety elements; the electrical outlet with
a ground pole.
3. A levelled and frm table designed for supporting the
weight of the cabinet and allowing the operator to work
comfortably. There must be free space for placing the
feet and its height must be adequate.
4. The floor on which it is located must be flat and
levelled.
5. The free space around the cabinet recommended by the
manufacturer must be respected. Likewise, the height
of the room must be verifed (the ceiling must be of
recommended height so that it can function without
hindrance).
6. Type B cabinets must have an extraction duct equipped
with the following required control devices: regulating
valves that allow the flow of air to be isolated and
regulated.
7. Gas connections must be in the immediate vicinity of
the cabinet in order to facilitate the connection to these
service valves.
8. The cabinet must be certifed annually to verify that it
complies with the established requirements in the NSF
49 Regulation.
USE OF THE SAFETY CABINET
Correct utilization of the biological safety cabinet is achieved
by complying with the following instructions:
1. Plan the work to be done in the biological safety cabinet
in advance. Determine what procedure and equipment
will be used. Coordinate the time of the cabinets use
with the other laboratory professionals in order to avoid
interruption or undesired traf c while it is in use.
2. Turn on the cabinet. Turn of the UV lamp if lit. Turn on
the fuorescent light lamp and the cabinets ventilator.
Verify that the grids in front and behind are free of
obstructions. Prepare the work area. Allow the cabinet
to function for at least 15 minutes.
3. Wash hands and forearms with germicidal soap. Put on
the personal protective apparel: coat/overall with long
sleeves and adjustable cufs, protective eyeglasses and
mask if the work requires it. Prepare the interior surfaces
of the cabinet applying 70% ethanol or a suitable
disinfectant. After this, let the air fow through.
4. Only load and install the materials and equipment
required for the test or manipulation. Distinguish
between the clean areas and dirty areas. Place the
material in such a way that the clean materials do not
mix or cross used or dirty materials or impede the
circulation of the internal air through the front and
back grids. Place a biosafety bag for disposing waste
materials, a container with disinfectant for the pipettes
and a container for storing sharps. Avoid locating very
large objects near one another. Upon fnalizing the
placing of the materials, the fow of air must be allowed
to sweep through the cabinet for approximately 3 to 5
minutes in order to eliminate any particle produced or
freed during the loading of materials and equipment.
5. Initiate activities. Slowly introduce hands into the work
area. Carry on the processes and tasks in a methodical
and careful manner (from the clean areas to the
1
The Laboratory Biosafety Guidelines, 3rd. Edition-Draft, Health Canada, 2001.
CHAPTER 6 BI OLOGI CAL SAFET Y CABI NET
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potentially contaminated areas). Keep the materials
at least 10 cm behind the front grid. Try to perform
the most risky and contaminating activities towards
the back of the cabinets work area. Avoid the use of
open fames of lighters since this breaks the laminar
fow pattern and may burn the flter. Avoid removing
hands from the work area until all procedures are
accomplished and the potentially dangerous materials
are disposed of in the biosafety bag or in the pipette
and sharp containers.
6. Clean the cabinet, allowing the air to fow freely for 3 to
5 minutes upon ending all the procedures.
7. Decontaminate the surfaces of all the materials
and equipment in contact with the biologically
contaminated material. Apply 70% ethanol or a suitable
disinfectant and allow drying. Lift the equipment and
materials and disinfect the area underneath. Cover the
open containers before removal from the work area.
Transfer materials to their appropriate place (incubator,
autoclave, etc.).
8. Discard the gloves and remove personal protective
elements. Dispose of these following the laboratorys
established procedure. Wash hands with a lot of water
and soap.
9. Turn of the ventilator, the fuorescent lamp, close the
front opening and turn on the ultraviolet light.
Note: In case of a leak or spill inside the cabinet while in
use, it must be kept in operation and all the objects or
equipment involved must undergo a process of surface
decontamination. This will prevent the cabinet from
releasing contaminants.
Decontamination of the cabinet
The decontamination of the biological safety cabinet is an
activity which must be done before any maintenance work
involving opening its surfaces or internal components.
Whenever any of the processes indicated next are needed,
decontamination of the cabinet must be done previously.
1. Changing of flters.
2. Conducting tests requiring access to the interior surfaces
or exposure of the cabinet.
3. Before conducting certifcation tests when the cabinet
has been used with classifed agents such as level 2 or
3 biological risk agents.
4. Before moving the cabinet to a diferent location.
5. After a spill of a material containing high risk agents.
The most suitable decontamination procedure must be
defined by the professional responsible for industrial
safety and professional risks. In annex G of the NSF 49
Standard, the procedure for decontaminating the cabinet
using depolymerised paraformaldehyde is described. Only
professionals who have received the relevant training must
conduct such procedures.
ROUTINE MAINTENANCE
Warning: The maintenance of internal components must
only be done by trained and qualifed personnel. In order
to carry out maintenance on the internal components,
decontamination must be done previously. Personal
protection must be worn to perform the routines.
General maintenance required for the biological safety
cabinet is for the most part simple to perform. The routines
and frequencies are shown below:
Frequency: Weekly
1. Decontaminate the work surface and the interior
surfaces of the cabinet with 70% ethanol.
2. Clean the front glass door and the surface of the
ultraviolet lamp, using a domestic cleaning solution.
3. Verify the precision of the manometers reading,
indicating any fall in pressure fowing through the HEPA
flter. Register the date and the reading in the cabinets
log book.
Frequency: Monthly
1. Clean the exterior surfaces, especially the front and
the upper part using a piece of damp cloth in order to
remove the dust.
2. Disinfect the surface of the lower compartment with
70% Ethanol or a suitable disinfecting solution.
3. Verify the state of the service valves.
4. Do the tasks due on a weekly basis.
Frequency: Annually
1. Carry out the certification process according to
established outlines in the NSF 49 regulation.
2. Check the intensity of the UV lamp
1
with a radiometer.
Substitute it if necessary.
3. Test the state of the fuorescent lamp. Substitute it if
necessary.
4. Perform the tasks due on a monthly basis.
Removal of the work surface
For the removal of the work surface the following procedure
is required:
1. Decontaminate the surface before removing it.
2. Loosen and remove the attachment screws located on
the front part of the work surface.
3. Loosen, but do not remove the attachment screws
located on the back part.
4. Raise the front end and remove it, pulling it towards the
front part of the cabinet.
5. Decontaminate the interior part of the work surface.
6. To assemble it, perform the activities described in steps
2, 3 and 4 in reverse order.
1
UV lamps have irradiation capacity lasting approximately 7,500 hours. Some
manufacturers suggest annual substitution.
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
41
Changing of the ultraviolet lamp
In order to change the ultraviolet lamp, the manufacturers
instructions must be followed. In general, the following
procedures are done:
1. Turn on the cabinet and leave it working for 5
minutes.
2. Raise the front window to its maximum position.
3. Decontaminate the interior surfaces and the UV lamp.
4. Disconnect the electrical feed to the cabinet.
5. Disconnect the UV tube from its connectors turning
it 90 degrees. Next, install a spare part with the same
characteristics as the original. Some manufacturers have
installed the lamps on a plate located in the front of the
cabinet, which is necessary to unscrew and lift so that
the assembly of the lamp is kept visible. Once this is
done, the lamp can be substituted as indicated above.
Specialized maintenance
Eventually, the cabinet will require specialized maintenance.
The following are some procedures to be done according
to the manufacturers technical service manuals by a
specialized contractor.
1. Annual certifcation in accordance with Regulation NSF
49 outlines.
2. Motor change. Generally, it uses maintenance-free sealed
rollers and function by induction through frequency
control. This motor does not have brushes. (*)
1
.
3. Replacing ventilators. (*)
4. Replacing the HEPA flter (*). The replacement frequency
depends on the use of the cabinet and the system of
environmental control installed in the laboratory. If
there is a good control of dust, the flter could last many
years.
5. Repair of the electronic control system: fow control
alarms, position of the window, velocity controls.
6. Repair/cleaning of the fow regulator valves, bell type
adjustment fttings.
Cabinet certication
The certifcation process of the biological safety cabinets
is regulated by Standard NSF 49, which applies to all Class
II cabinets. This defines materials, design criteria and
construction, operation parameters and tests which allow
the cabinet to be guaranteed as safe and suitable for the
work performed. The following is a list of tests, in which
standards mentioned are included. The standards must be
consulted for details. The certifcation process comprises
the following tests:
1. Air tightness test. This is done on the exterior surfaces.
Determine if joints, seals, penetration and solderings are
free from leaks.
2. HEPA lter leak tests. Determines the integrity of the
supply and extraction of HEPA flters, their location and
mounted frames.
3. Temperature increase test. Determines the maximum
temperature increase in the cabinet when the ventilator
and lights are operating.
4. Noise test. Determines the level of noise produced by
the cabinet.
5. Luminous intensity test. Determines the luminous
intensity on the cabinets work surface.
6. Vibrations test. Determine how much vibration there
is in the cabinet when it is functioning.
7. Protection test to personnel, to the product and cross
contamination biological tests. The test determines
if aerosols are contained in the cabinet, if external
contaminants reach the work table area and if aerosols
are reduced by the cabinet.
8. Stability test. Determines if the cabinet has structural
stability. Analyzes the resistance to shaking, to distortion
by means of applied force, to defection of the work
surface subjected to load and resistance to the tilting
of the work surface due to heavy loading conditions.
9. Vertical ow velocity test. Determines the velocity of
the air moved vertically towards the work surface.
10. Entry ow velocity test. Determines the velocity at
which the fow enters the cabinet through the front
opening and the cabinets extraction volume.
11. Smoke test. Determines if the fow of air along the
entire perimeter of the front opening advances towards
the cabinet, and if the vertical fow moving towards the
bottom does not show dead points or fow backs on the
work surface.
12. Drainage escape test. Defnes the contention capacity
for spills below the work surface.
13. Motor/ventilator system functioning test. Determines
if the system provides the necessary static pressure.
14. Electric system test. Determines if there are potential
risks of electrical discharges. Measures the escaping
currents, the polarity, the functioning of the ground
defect protection system and the ground circuit
resistance.
FUNCTIONAL EVALUATION (ALTERNATIVE)
In case there are biological safety cabinets in the laboratory,
but no authorized certification services available, the
personnel responsible for maintenance has the option of
conducting annual revision procedures based on Standard
NSF 49. Duly documented, it should identify with low levels
of uncertainty if the cabinet is in good condition and its
operation normal
2
. The following are outlines of how these
activities must be done.
1. Installation evaluation. Verify that the cabinet
installation conditions are in accordance with the
recommendations from the manufacturer.
1
(*) These require specialized decontamination beforehand.
2
The functional evaluation is essentially based on the availability
(institutional or zonal) of properly trained and experienced technicians and
engineers.
CHAPTER 6 BI OLOGI CAL SAFET Y CABI NET
42
2. Operational evaluation. Test to see if the cabinet is
working in accordance with its manufacturing and
design characteristics.
3. Performance evaluation. Verify the cabinets capacity to
provide an adequate work space in normal and critical
working conditions.
In the following table are featured the parameters to be
taken into account in the functional evaluation. These are
generally included in inspection forms
1
designed for this
purpose.
Parameters Observation
Institutional identifcation of cabinets Brand, model, type, series, location, inventory code, date.
ELECTRICAL
Voltage Voltage measurement. Requires a voltmeter.
Amperage Amperage measurement. Requires a voltmeter or amperemeter clip.
Motor/ventilator Verifcation of operation temperature. Verify noise level and vibration.
Illumination Fluorescent Confrmation that the lamp is functional.
Illumination Ultraviolet Confrmation of the operational hours of the lamps and their light intensity. Requires a radiometer.
Electrical outlet Integrity revision, quality of the contact and available voltages.
Switches Control of state and integrity.
Integrity cables and connectors Visual verifcation.
Alarms Testing of state and calibration.
PHYSICAL
Internal/external fnishes Visual verifcation.
State of flters and pre-flters Visual verifcation. There must be no leaks, neither in the fltering material nor in the seals.
Seals/gaskets Visual verifcation. There must be no leaks.
Sliding window Visual verifcation. Must be able to be moved smoothly and maintain the selected positions.
OPERATIONAL
Flow velocity Control of velocity according to the class and type of cabinet. Requires an anemometer (wind gauge).
Noise level Requires audiometer.
Pressure diferential in the HEPA flter. Take a manometer reading of the cabinet.
PERFORMANCE
Counting of particles Method defned in the Federal Standard 209D, E. Requires DOP generator, photometer and particle counter.
CONDITIONS OF THE INSTALLATION AREA
Temperature Requires thermometer: approximately 2022 C.
Humidity Requires hygrometer: approximately 4555 %.
Cleanliness Must be adequate.
Air currents There must be no air currents to afect the working of the cabinet.
Table of functional evaluation of biological safety cabinets
1
Each institution designs its own formats for record keeping of technical
maintenance.
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
43
TROUBLESHOOTING TABLE
1
PROBLEM PROBABLE CAUSE SOLUTION
Neither the light nor the ventilation system in the
cabinet works.
The cabinet is disconnected from the electrical
outlet.
Verify that the cabinet is connected to an electrical
outlet and that the cable is well connected to the
cabinets electrical box.
There is no electrical feed in the connection. Confrm that the electrical outlet is energized and
that the circuit breaker is not deactivated (thermo
magnetic protection). Restart switches.
The cabinets ventilator is functioning but the light
does not.
The lamp is defective. Replace the lamp. Use one with the same
characteristics of the original
The lamp is badly connected. Check the lamps connection. Adjust to the correct
position.
The thermo magnetic protection of the service
breaker is activated.
Reconnect the circuit breaker.
The lamps wire is disconnected. Check the lamps wire.
The lamps ballast is defective. Replace the ballast.
The ventilator is not blowing but the light is coming
on.
The front window is closed. Open the window until it reaches the work position.
The ventilators motor is defective. Replace the motor ventilator set.
The ventilators motor is disconnected. Check the motors connections.
The manometer indicates an increase in the fall of
pressure through the flter.
Retention of particles in the HEPA flter has
increased.
Normal process during the useful life of the flter.
There is blockage in the grids or return slots. Verify that the grids are not obstructed by
equipment or material.
The extraction pipe is obstructed. Test that there are no existing blockages or
restrictions in the extraction pipe.
There is a blockage or restriction under the work
surface.
Verify that the pipe below the work surface is free of
obstructions.
There is contamination in the samples manipulated
in the cabinet.
Work procedures are inadequate. Check that the cabinet is being used according to
procedures and good practices.
Restrictions in the return slots or blockage of the
extraction duct.
Test the return and extraction system to see if they
are free from obstructions.
The cabinets external factors afect its fow patterns
on the inside and cause contamination.
Verify the installation of the cabinet and the
procedures that are being carried out.
The HEPA flter is defective. Replace the HEPA flter and certify the cabinet.
1
Purifer Delta Series, Biological Safety Cabinets, Users Manual, Kansas City, Labconco Corporation, Part N 36960-20, Rev. A ECO B296.
CHAPTER 6 BI OLOGI CAL SAFET Y CABI NET
44
BASIC DEFINITIONS
Aerosol. A suspension of fne solid or liquid particles in the air. Their average diameter ranges between 10
-4
and 10
-7
cm.
Air supply. Air which enters the cabinet through the front or work opening and replaces the air extracted from the cabinet.
Biological Safety cabinet. Equipment with appropriate ventilation conditions protecting the user, the environment and the sample from aerosols and microparticles,
associated with the management of potentially infectious biological material in laboratories as a result of activities such as agitation, centrifugation, use of pipettes
and opening of pressurized containers.
Certifcation. Procedure establishing that the biological safety cabinets functioning complies with criteria and minimum requirements to operate safely. Standard
NSF 49 applies to the Class II cabins, Type A, B1, B2 and B3.
Decontamination. Removal or destruction of infectious agents; removal or neutralization of toxic agents.
HEPA flter. A flter with the ability to remove particles with average diameters of 0.3 m with 99.97 % ef ciency. These flters are constructed of Boron silicate
micro fbres bonded together with a water resistant adhesive. The fltering material is folded inside of a frame with the aim of increasing the fltration area.
Laminar fow. Non-turbulent fow of a viscous fuid (e.g. air) in layers near a boundary. It occurs when Reynolds number [Re] is less than 3000.
NSF. An acronym of the National Sanitation Foundation, a non-proft organization dedicated to research, education and service, which seeks to resolve problems
related to human beings, promote health and enrichment of the quality of life through conservation and improvement of the environment. NSF standards supply
the basic criteria for promoting salubrious conditions and public health protection.
Toxic. A substance with a physiologically adverse efect on the biological systems.
Ultraviolet light (UV). This is electromagnetic radiation, the wavelength of which is between 200 and 390 nm. It is used in biological safety cabinets for its
germicidal properties.
Work surface. A surface used when performing work, operation or activity inside the biological safety cabinet in this case.
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
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Chapter 7
Centrifuge
GMDN Code 15115 10778 10778
ECRI Code 15-115 15-117 15-116
Denomination Centrifuges, standing, low velocity,
non-refrigerated, for blood bank
Centrifuge, standing,
refrigerated
Standing centrifuge
The word centrifuge comes from the Latin word centrum
which means centre and fugere which means to escape. The
centrifuge is designed to use the centrifugal force generated
in rotational movements to separate the constitutive
elements of a mixture. There is a wide range of centrifuges
capable of serving specifc industry and research needs. This
chapter focuses on standing centrifuges normally used in
public health and clinical laboratories.
PHOTOGRAPH OF CENTRIFUGE
PURPOSE OF THE CENTRIFUGE
The centrifuge uses centrifugal force (the force generated
when an object rotates around a single point), for separating
solids suspended in a liquid by sedimentation, or liquids of
diverse density. The rotational movements allow forces much
greater than gravity to be generated in controlled periods
of time. In the laboratory, centrifuges are generally used in
processes such as the separation of solid components from
biological liquids through sedimentation and in particular
of blood components: red cells, white cells, platelets among
others and for conducting multiple tests and treatments.
There are several kinds of centrifuges. The most widely used
in public health, surveillance and clinical laboratories are the
table-top centrifuge, the ultracentrifuge, the haeamatocrit
centrifuge and the standing centrifuge.
OPERATION PRINCIPLES
Centrifuges represent a practical application of Newtons
law of motion. When a body of mass [m] turns around
a central point [O], it is subjected to a centripetal force
[N] directed towards the rotation axis with a magnitude
N = m
2
R, where [m] is the mass of the body, [R] is the radius
and is the angular speed. Centrifuges possess a rotating
axis on which is mounted a rotor with sample receiving
compartments. Tangential speed is defned by the following
equation: VT=R.
P
h
o
t
o

c
o
u
r
t
e
s
y

o
f

B
e
c
k
m
a
n

C
o
u
l
t
e
r
CHAPTER 7 CENTRI FUGE
46
When the system spins at a speed of radians per second,
the samples are subjected to the centrifugal force Fp of the
same magnitude as N, but in an opposite direction. The
fgure shown below
1
features a diagram of the concept,
of its actual application and of the obtained result. This
Fp force acts on particles in the substance centrifuged,
causing them to separate as a result of diferences in density.
Denser particles will settle at the bottom of the tube in
shorter periods of time, while lighter ones require longer
periods of time, settling onto those of greater density. The
relationship between the centrifugal acceleration [
2
r ] to a
given radius [r] and the force of gravity [g] is known as the
relative centrifugal feld or [RCF]
2
.
RCF =
r
2
g
The RCF is the tool which allows rotors of different
specifcations to be compared when equivalent centrifugal
efects are required.
COMPONENTS OF THE CENTRIFUGE
The most important components of a centrifuge are the
following
3
:
The electric/electronic control which generally has the
following elements:
1. On and off control, operation time control (timer),
rotation speed control (in some centrifuges), temperature
control (in refrigerated centrifuges), vibration control
(safety mechanism) and brake system.
2. Refrigeration system (in refrigerated centrifuges).
3. Vacuum system (in ultracentrifuges, not shown in the
fgure).
4. Base
5. Lid/cover
6. Casing
7. Electric motor
8. Rotor. There are different types of rotors. The most
common are the fxed angle, the swinging buckets, the
vertical tube and the almost vertical tube types, which
are explained next.
1
Newtons law of movement, together with the explanation of the inertia
marks of reference can be consulted in books on physics, chapters on
uniform circular movement.
2
RCF. Relative Centrifugal Field.
3
The numbers identifying each component correspond to those in the
sectional diagram of the centrifuge.
Sectional diagram of a centrifuge (numbers correspond to
descriptions in the text above)
Figure 20. Centrifugal force concept
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
47
Types of rotors
Centrifuges use many diferent types of rotors. Among the
most commonly used are the following:
Type of rotor Characteristics Transversal cross-section
Fixed angle rotors. These are general purpose rotors. They keep tubes at a fxed
angle [] which by design, is specifed between 20 and 45
degrees. They are used for sediment sub-cellular particles.
The angle shortens the trajectory of the particles and the
centrifugation time compared to the swinging buckets
rotors.
Swinging buckets rotors. These are used for carrying out isopycnic studies (separation
by density) and rate-zonal studies (separation by
sedimentation coef cient), where maximum resolution of
the zones is required for the sample.
Vertical tube rotors. This type of rotor keeps tubes parallel to the rotational axis.
Thus, separate bands are formed across the tubes diameter,
not its length. These rotors are used for carrying out
isopycnic studies and in some cases, zonal limit separations
where a short centrifugation time is important. These rotors
use specially designed tubes.
Almost vertical tube rotors. This type of rotor is designed for gradient centrifugation
when some sample components do not participate in
the gradient. The small angle of these rotors reduces the
centrifugation time in comparison to fxed angle rotors.
Position in
Rotation
Position
at Rest
r
CHAPTER 7 CENTRI FUGE
48
Normally, manufacturers specify rotors to be used in
centrifuges by providing specialized publications of tables
with the following information:
1. Type of rotor. Specifes the type of rotor for which the
technical information is being provided.
2. Nominal capacity of the rotor. Defnes the capacity in
litres or litre submultiples. For example: 6 litres; 250 ml,
etc.
3. Maximum speed. This indicates the maximum speed
at which this particular rotor should be operated in
revolutions per minutes (RPM).
4. Maximum Relative Centrifugal eld (RCF) obtained by
that type of rotor.
5. k Factor, the sedimentation coef cient, defned by the
following equation:
Where:
= angular speed in radians per second
r
max
= maximum radius in mm, measured in the
centrifugation tube
r
min
= minimum radius in mm, measured in the
centrifugation tube
The time required for sedimentation can be calculated
in hours using this factor.
6. Information on the compatibility of the rotor with other
models of centrifuges from the same manufacturer.
Recently manufactured centrifuges have incorporated
numerous improvements into their design to provide
greater safety and longer operational life. Among advances
mentioned are controls based on microprocessors. By means
of software controlled by a keyboard, these have several
diferent operational programs in memory. According to the
type of rotor being used and procedure conducted, these
programs control the centrifugation time, the required
temperature, the rotors revolutions, the acceleration and
deceleration, alarms warning the operator about any
anomaly during operation.
Manufacturers have also incorporated induction motors
(without brushes) in centrifuges. These have the advantage
of electronically controlling currents and magnetic
fields regulating the rotors speed which reduces the
frequency of maintenance. Operation and maintenance
of such equipment must be carried out according to the
manufacturers recommendations.
INSTALLATION REQUIREMENTS
Centrifuges require the following for normal operation:
1. An electrical connection with a capacity suitable for the
equipment providing stable single phase or triphase
type voltage (depending on the model and specifcation
given by the manufacturer). In general, centrifuges use
110V or 220 V/60 Hz.
2. A clean, dust free environment with a frm levelled
foor.
3. If the centrifuge is refrigerated, it needs a free space on
the side of the condenser for adequate heat transfer.
4. A cabinet in which the centrifuge accessories such as
the alternate rotors can be kept.
ROUTINE MAINTENANCE
The routine maintenance required by a centrifuge depends
on multiple factors such as the incorporated technology,
usage intensity, training of users, quality of the electrical
feed and environmental conditions. The following are
general recommendations regarding adequate use and
most common maintenance for guaranteeing correct
operation. The routines or specialized repairs will depend on
manufacturers recommendations for each brand and model.
Always disinfect the rotor bowl, centrifuge head, buckets
and trunnion rings as applicable before any servicing of
centrifuges used to prepare clinical or infectious samples.
Priority recommendation. Verify that only qualifed personnel
trained and familiar with the use, care, risks and handling
of the centrifuge operates it. It is the laboratory directors
responsibility to supervise and take necessary precautions
so that personnel operating centrifuges understand the
implications of working with such equipment.
APPROPRIATE MANAGEMENT AND STORAGE
RECOMMENDATIONS
1
Rotors
1. Register the date of purchase of each one of the rotors,
including information related to the serial and model
number.
2. Read and understand the rotor manuals, equipment
and tubes before use. Comply with indications for use
and care specifed by the manufacturer.
3. Use rotors only in centrifuges for which these have
been manufactured. Do not interchange rotors without
verifying the compatibility with the centrifuge.
4. Register operation parameters for each rotor in a log
book in order to determine its remaining operational
life and to acquire its replacements when needed.
5. Use the recommendations regarding maximum speed
and sample density from the manufacturer. Each rotor
is designed for supporting a maximum level of efort;
these specifcations must be followed rigorously.
1
http://www.sunysb.edu/facilities/ehs/lab/cs.shtml
k =
ln r
max
r
min
( )

10
13
3600
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
49
6. Obey the recommendation related to reducing the
operation speed when working with high density
solutions in stainless steel tubes or plastic adaptors.
Manufacturers provide the related information.
7. Use titanium rotors if working with saline solutions
frequently.
8. Protect the rotors coating in order to avoid the metal
base from deteriorating. Do not use alkaline detergents
or cleaning solutions which can remove the protective
flm. The rotors generally made of aluminium [Al] are
covered by a flm of anodized aluminium which protects
their metal structure.
9. Use plastic brushes when cleaning the rotor. Metal
brushes scratch the protective coating and generate
sources for future corrosion. Corrosion is accelerated
in operation conditions and shortens the rotors
operational life.
10. If there are spills of corrosive substances, wash the rotor
immediately.
11. Air dry the rotor once cleaned and washed with water.
12. Store vertical tube rotors and almost vertical tube rotors
with the larger side facing downwards and without their
covers.
13. Store rotors in a dry area. Avoid leaving them in the
centrifuge.
14. Store swinging buckets rotors without the compartments
covers.
15. Lubricate spiral and O-rings, according to the
manufacturers recommendation.
16. Observe recommendations related to guaranteed times
and operational life of each type of rotor.
17. Avoid using rotors whose operational lives have
ended.
18. Use a shield if working with radioactive material.
19. Load or unload rotors inside a biological safety cabinet
if working with materials classifed as Biosafety level II
or higher.
20. Never try to open the cover of a centrifuge while it is
functioning and never try to stop the rotor by hand.
Tubes
Tube care includes aspects such as flling of the tubes,
adequate temperature selection, centrifugation speed
limitations, washing and sterilization. The principle
recommendations are the following:
1. Wash tubes, adaptors and other accessories by hand
using a 1:10 mild detergent solution in water and a soft
textured brush (not metallic). Avoid using automatic
dishwashers.
2. Avoid using alcohol and acetone since such liquids afect
the structure of the tubes. Manufacturers recommend
the solvent to be used with each type of centrifugation
tube material.
3. Avoid drying tubes in a drying oven. Dry always with a
stream of hot air.
4. Verify if the tubes are reusable or not. If they are
disposable, use them only once.
5. For sterilizing, it is necessary to verify the material from
which the tube is made, as not all can stand sterilization
by heat. Glass tubes are normally sterilized with vapour
at 121 C for 30 minutes.
6. Store tubes and bottles in a dark, fresh, dry place,
far from chemical vapours or ultraviolet radiation
sources.
7. Verify maximum flling levels and the sealing of thin
wall tubes in order to avoid collapse inside the rotor
by the action of the centrifugal force. Comply with
manufacturers recommendations.
Preventive maintenance
Warning: Never carry out a technical intervention in a
centrifuge if it has not been previously decontaminated.
The most important maintenance routines performed on a
centrifuge are the following:
Frequency: Monthly
1. Verify that the centrifuge external components are free
of dust and stains. Avoid afecting the rotor with spills.
Clean the rotor compartment using a mild detergent.
2. Test that the rotors connecting and adjustment
mechanisms are in good condition. Keep the points
lubricated as the manufacturer recommends.
3. Verify the locking /safety mechanism of the centrifuges
cover. This is fundamental in guaranteeing operators
safety as this mechanism keeps the cover of the
centrifuge closed while the rotor is turning.
4. Check the lubrication state of elements such as for
O-rings as the manufacturer recommends. Always use
lubricants according to the manufacturers instructions
(frequency and type of lubricants). In recently
manufactured centrifuges, there are sealed ball bearings
which do not require lubrication.
5. Verify the state of gaskets and watertight joints.
Frequency: Annually
1. Verify that electronic cards are clean and well connected.
2. Test operation controls needed for selection of the
diferent parameters of the centrifuge: speed, time,
temperature, alarms selectors and analogous or digital
instruments.
3. Verify compliance with electrical standards. Use an
electric safety analyzer: earth resistance test, escaping
current test.
4. If the centrifuge is refrigerated, test the temperature by
using an electronic thermometer. The temperature must
not vary by more than 3 C.
5. Examine the exactitude of the time controls. Use a timer.
The time measured must not vary by more than 10 %
of the programmed time.
CHAPTER 7 CENTRI FUGE
50
6. Verify the actual rotation speed against the selected
one using a normal load. The testing is done with
a tachometer or a photo tachometer. If the hatch
is not transparent, the procedure indicated by the
manufacturer must be followed.
7. Confrm the functioning of the brake system.
8. Verify the functioning of the refrigeration system in
refrigerated centrifuges. The following are the most
important activities:
a) Check the selected temperatures. These should
not vary by more than 3 C from the temperatures
measured on the digital thermometer.
b) Verify the state of the air intake filter. If the
flter is obstructed, clean or substitute with an
equivalent.
c) Conduct a detailed cleaning of the difusing wing of
the condenser to eliminate the flth deposited. This
maintains the heat transference rate according to
the design specifcations. If abnormal functioning
is detected, seek assistance from a specialized
service technician.
Note: Avoid spilling liquids on control keys. The keys must
be operated with the fngertips: The operator should avoid
using fngernails, as this can result in the perforation of their
protective membrane.
Every six months:
Verify the state of the motors brushes, if the centrifuge has
a motor with brushes. Substitute with new ones (with the
same specifcations as the original) if necessary. Perform this
routine every six months.
Tools and required instrumentation
In order to carry out the maintenance inspections normally
required for a centrifuge, the following tools or instruments
are necessary:
1. A key for tightening and slackening the rotors nuts.
2. An electrical safety analyzer or an instrument for
measuring escaping current.
3. A timer.
4. An electronic thermometer with exactitude of 0.5C for
refrigerated centrifuges.
5. A tachometer or photo tachometer.
TROUBLESHOOTING TABLE
Rotors
1
PROBLEM PROBABLE CAUSE SOLUTION
Severe vibration. The rotor is unbalanced. Balance the rotors load. Fill all the opposite tubes
with the same level of liquid of same density.
Distribute the weight of the opposite tubes
symmetrically.
Load fxed angle or vertical tube rotors
symmetrically.
The speed selected is near the rotors critical speed
range.
Select a rotation outside of the critical speed range.
The rotor is incorrectly mounted. Verify the rotors assembly. Test that it is well
adjusted.
There is a lack of lubrication in the rotors supports. Lubricate the pivoting axis according to the
manufacturers recommendation. For e.g. each 250
centrifugation procedures.
Rotor covers, canister or cubes dif cult to loosen
after centrifugation.
A vacuum is being produced during centrifugation. Open the ventilation line in the upper part of the
rotor or bucket to eliminate the vacuum.
The rings are contaminated with flth, dried
lubricants or metallic particles.
Perform routine cleaning of the rings and lubricate.
Use recommended products recommended by the
manufacturers.
1
Rotors and Tubes for Beckman Coulter J2, J6 and Avanti J series centrifuges, Users Manual, Palo Alto, California, The Spinco Business Center of Beckman Coulter, 2001.
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
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Tubes
PROBLEM PROBABLE CAUSE SOLUTION
The tubes leak. The covers are badly secured. Adjust the covers.
The tubes are too full. The meniscus must be lower in order to prevent
leaks.
The maximum recommended level has been
exceeded in the open tubes.
Verify the volume and speed recommendations for
the centrifugation.
A defcient seal is presumed in the rapid seal tubes. Press lightly, after heat sealing (only if the contents
are not afected). If leaks are visible, seal again.
The tubes are cracked or broken. The tubes can be broken or become fragile if they are
used below the recommended temperature.
If the sample is frozen, warm to 2 C before
centrifuging. Evaluate how the tubes behave at low
temperatures before centrifuging.
The tubes become fragile with age and use. Discard expired tubes, use new ones.
Various systems
PROBLEM PROBABLE CAUSE SOLUTION
The main switch is in the on position but the
centrifuge is not functioning.
There is no power to the instrument. Verify the power supply.
The centrifugue cover cannot be opened. The centrifuge is of. Turn the centrifuge ON. Press the handle and open
the cover.
The balance indicator is activated. The load to be centrifuged is unbalanced. Balance the load to centrifuge.
The centrifuge is not levelled. Level the centrifuge.
There is a vibration at low speed. The rotor adjustment mechanism is slack. Correctly adjust the fastening system.
The load is unbalanced. Verify the balance of the load to be centrifuged.
The selected speed is close to the rotors resonance
point.
Select a more elevated rotation speed or use a
diferent type of rotor.
There are fuctuations in the rotation speed. The transmission belts are in a bad condition (*). Turn of the centrifuge. Verify the condition and state
of the belts. The belts must be tempered.
The rotation speed does not reach the selected
speed.
The brushes are defective. Turn of the centrifuge. Verify the condition of the
brushes. If this is the problem, put new brushes with
the same specifcations as the originals.
The speed control calibration is maladjusted. Adjust the speed control calibration.
The chamber is cold but the rotor is warm. The temperature is incorrectly selected. Verify the temperature selection.
The display which signals the state of the brushes
is on.
The brushes are in a bad condition. Turn of the centrifuge. Verify the condition of the
brushes. Substitute the brushes by others with the
same specifcation.
(*) Valid procedure in centrifuges with potential belt transmission system.
CHAPTER 7 CENTRI FUGE
52
BASIC DEFINITIONS
Anodized coating. A hard, thin layer of aluminium oxide, which is deposited on the surface of a rotor by means of electrochemical processes with the aim of
preventing corrosion. The coating is often fnished in various colours.
Angular speed. The turning rate of a body measured in radians per second. It is calculated using the following formula:
Where:
rpm = revolutions per minute
= constant with a value of 3.1416
Brush. A device that transmits electrical energy between the external electrical connection (cables in a static state) and the internal components (in rotation) of a
motor. In general, brushes are manufactured in very soft textured graphite and, in motors, must be changed regularly (every six months).
Centrifugal force. Apparent force equal and opposite to the centripetal force, driving a rotating body away from the centre of rotation and caused by the inertia of
the body. It is one of the components of the inertia vector, which equals the set of forces acting on a body. Its magnitude is always [m x a
n
] and its direction radial,
moving away from the centre.
Density. A bodys mass by volume unit, generally expressed in gram per cm
3
.
Isopycnic separation. A method for separating particles based on the density of the particles fotation. It is known as sedimentation in balance. The speed of a
particle due to diferences in density is given in the formula:
Where:
v = speed of sedimentation
d = diameter of the particle

p
= density of the particle

c
= density of the solution
= viscosity of the liquid medium
g = gravitational force
Radian. A unit of angular measure equal to the angle subtended at the centre of a circle by an arc equal in length to the radius of the circle. It is expressed as the
ratio between the arc formed by the angle with its vertex in the centre of the circle, and the radius of that circle.
RCF (Relative centrifugal feld or force). A relationship between the centrifugal acceleration and a specifc speed and radius, [r
2
] given with the normal gravity
acceleration. It is calculated by means of the following equation:
Where:
R = radius in mm
= angular speed in radians per second
g = Standard gravity acceleration = 9 807 mm/s
2

Resonance. A situation in which a mechanical system vibrates as a response to a force applied at the systems natural frequency.
Sedimentation. Particles from a suspension settling at the bottom of the liquid as a result of the action of the gravitational force. During centrifugation, this process
is accelerated and particles move away from the rotational axis.
=
2 rpm
60
D =
m
V
v =
d
2

p

c ( )
18








g
dr
dt






=
2 rpm
60
RCF =
r
2
g
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Chapter 8
Water Distiller
The word distiller comes from the Latin word distillare
which means to vaporize liquids through heat. The water
distiller, also called distillation unit or water still, used in the
laboratory, purifes running water by means of controlled
vaporization and cooling processes. Upon applying thermal
energy to water in a liquid phase by a warming process, it
is changed into vapour. This allows the water molecules to
separate from the molecules of other substances mixed
or diluted. The water vapour is collected and passed
through a condenser, where it is cooled and returned to
the liquid phase. Then, the condensed water is collected
into a diferent storage tank. Distilled water shows pure
characteristics compared to running water; it is practically
free of contaminating substances.
PURPOSE OF THE WATER DISTILLER
The water distiller facilitates obtaining very pure water from
potable water normally provided by the aqueduct services
in urban centres. Distilled water is characterized by a lack
of solids in suspension. It is used in multiple applications
in centres which provide health services, especially in
laboratory units, in washing, sterilization and dietetics. The
more specialized the procedures are in the laboratory, the
greater will be the level of purity required. For example: the
preparation of reagents or biological material requires water
of the highest quality. Distillation is one of the fundamental
processes to achieve this (although it may not be the only
one required). Water used in laboratories must be free of
pyrogens, with a concentration of total solids no greater
than 1 ppm, a pH value between 5.4 and 7.2 and an electrical
resistance of at least 3 x 10
5
ohm/cm at 25 C
1
.
1
Warming cabinets, sterilizers, and associated equipment, Division 11
Equipment, USACE/NAVFAC/ AFCESA, UFGS-11710, July 2003.
DIAGRAM OF A WATER DISTILLER
GMDN Code 40478
ECRI Code 15-136
Denomination
Distillation units
1. Vapour Generator
2. Water Level Gauge
3. Control Valve
4. Hydraulic Connection
5. Water Liquid Phase
6. Immersion Resistance
7. Cooling Water Exit
8. Condenser/Distiller
9. Activated Carbon Filter
10. Distilled Water Deposit
11. Cold Water Entry
Figure 21. Water distiller
CHAPTER 8 WATER DI STI LLER
54
OPERATION PRINCIPLES
The function of a distiller is based on a phenomenon
demonstrated in nature known as the water cycle. The
energy coming from the sun heats the water from the seas
and transforms part of it into water vapour. This vapour is
concentrated in clouds. When atmospheric conditions are
suitable, these cool and condense the water which returns
to the surface of the Earth in the form of rain.
Functioning of the water distiller
The water distiller reproduces the natural phenomenon
described above. The configuration and design vary
depending on the volume of water required. The following
is a general explanation of the components of a distiller and
a description of how these function.
1. Vapour generator. Also known as the boiling tank,
this component is the container where the water to
be distilled is stored. In general, it has a hydraulic
connection which allows the water evaporated and
distilled to be replenished. It is generally made of glass
in small distillers or of stainless steel with copper, tin or
titanium coverings in large capacity machines. It can
have level, fow and water quality feed controls, which
protect the distiller in case some irregularity in the
water supply occurs. As a source of energy, it uses the
water vapour coming from a boiler or vapour generator,
or the thermal energy from electrical immersion
resistors through direct conduction. These cause the
water temperature to rise until, in normal conditions
(atmospheric pressure equal to an atmosphere and
gravity acceleration equal to 9.80665 m/s
2
) water in the
liquid phase is transformed into vapour at 100 C.
2. Water level. Device which allows the quantity of water
to be regulated inside the vapour generator. It is joined
directly to the connection which supplies the water
used by the distiller. When the quantity of water in
liquid phase contained in the boiling tank decreases,
the device allows the quantity of liquid evaporated to
be recovered.
3. Control valve. Mechanical or electromechanical device
which allows the fow of water towards the vapour
generator tank to be regulated.
4. Hydraulic connection. Network which supplies water
in liquid phase to the vapour generator tank.
5. Water in liquid phase. Water inside the vapour generator
tank. It receives thermal energy from the immersion
resistors and it is converted to vapour when the required
temperature and pressure conditions are met.
6. Immersion resistors. Devices generating heat when
an electrical current circulates through them. These
are isolated by a ceramic cap and protected from the
external environment by a metal shield.
7. Refrigeration water outlet. Line carrying the water
used for condensing the water vapour thus removing
the thermal energy from it (cooling).
8. Condenser. Device in which the vapour loses thermal
energy, cools and returns to its liquid phase. In order
to accelerate the process, forced convection by low
temperature fuid circulation (air or water) around the
line through which the vapour fows is used.
9. Filter. Distillers have activated carbon flters located at
the exit of the condenser or collector. These eliminate
favours or particles which may be present in the vapour
being condensed.
10. Distilled water container. Device in which the fuid
completing the distillation process is collected. Distilled
water must be stored in special plastic containers to avoid
ionic contamination. Polyethylene, polypropylene or
polytetrafuoroethylene containers are generally used.
INSTALLATION REQUIREMENTS
Depending on the design, capacity and type of distiller,
the required installation may vary. The most common
requirements are the following:
1. A well ventilated environment in which the equipment
can be installed. This is necessary because the distiller
transfers heat to a fuid and increases the temperature
of the area where it is installed. It is necessary to leave
free space around the distiller so that the fow of air is
facilitated. Some distillers are assembled inside a metal
box and need to be installed on a support to facilitate
the circulation of air under them.
2. A potable water connection. Typically the required
hydraulic connection has a diameter of 1/2. To ensure a
smooth operation, the quality of the water feeding the
distiller must be evaluated to determine if it is necessary
to install a treatment system
1
to prevent the presence
of incrustations or sediments in the vapour generating
tank and on immersion resistors. Potable water is used
for feeding the vapour generator and for refrigerating
the condenser
2
.
3. A distilled water connection. The distilled water produced
is initially collected into a storage tank. In large capacity
equipment, it is distributed to consumption points from
the tank by means of a network. In small or medium
equipment, it is transferred to containers from which it
is used at the feed points.
4. Cleaning connection. This is used to drain impurities
which may accumulate in the vapour generator tank
using a siphon located near the distiller.
1
Water treatment has been designed for removing substances normally
present in water due to the great solvent capacity of water. The substances
in general are inorganic ions (anions and cations) such as bicarbonate,
sulphite, chloride, calcium, magnesium, sodium, potassium, magnesium,
iron, nitrates and traces of many others.
2
Some manufacturers cool the condenser through the use of ventilators
which make air circulate on the condensers fns, generating heat
transference processes by forced convection from the difusion surface to
the environment.
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5. An electrical connection equipped with control
and safety devices complying with the national and
international electrical standards used in the laboratory,
adapted to the capacity of the resistive elements of the
distiller. In general, the voltage is 220-240 V, 50/60 Hz.
Note: Always verify manufacturers recommendations on
installation to ensure the distiller is operating according to
the specifcations.
ROUTINE MAINTENANCE
The maintenance depends on the design and capacity of
the distiller. The maintenance described in this manual
focuses on a distiller equipped with a stainless steel vapour
generator tank with immersion resistors and a condenser
refrigerated through a ventilator impelling air (on or through
the condensers difusing fns).
Warning: Before carrying out an inspection or routine
maintenance, verify that the distiller is turned off and
disconnected from the electrical source.
Inspection and cleaning of the vapour generator tank
Frequency: Monthly
1. Remove the protective panel or open the door allowing
access to the boiling tank or vapour generator.
2. Remove the cover of the boiling tank.
3. Visually verify if the interior walls or the immersion
resistors show solid deposits or sediments. The quantity
of deposits present depends on the quality of water fed
to the distiller. If there is an accumulation of sediments,
it must be cleaned to avoid damaging the resistors
1
.
4. Clean accumulated deposits. In general, the cleaning
process requires a chemical product especially designed
for removing them. The product must be selected
according to the characteristics of the water used. This
is determined by a chemical analysis.
5. Drain water from the generator tank until its level is
approximately 10 cm above the location of the water
level probe or the immersion resistance (verify that the
water level is higher than the base of the tank to ensure
that all of the elements stay submerged in water).
6. Add the chemical product recommended for the type
of water used.
7. Mix well.
8. Allow the chemical to act overnight or as recommended
by the manufacturer.
9. Drain the contents of the tank on the following
morning.
10. Add clean water, wash and drain until the chemical
has been completely removed along with the mineral
residues from the afected surfaces.
11. Reinstall the cover.
12. Place the front panels or adjust the door.
13. Operate the equipment normally.
Warning: Under no circumstances, should the solution used
for removing sediments be distilled.
Change of the activated carbon lter
Frequency: Every three months
Normally, the activated carbon flter is submerged in water
below the dispenser system which comes from the distilled
water storage tank. It is assembled on a casing installed on
the distilled water distribution line. In general, it is a device
which can be easily substituted. The following process is
generally done:
1. Unscrew the top of the flter.
2. Remove the used fltering element.
3. Install a new element with the same characteristics as
the original.
4. Reinstall the top of the flter.
Warning: The flter is adjusted inside its casing by means
of O-rings or gaskets that must be installed carefully within
their grooves in order to avoid leaks of distilled water.
Cleaning of the condenser
Frequency: Annually
1. In order to clean the condenser, it is necessary to remove
the protective panels or open the door, giving access to
the condenser.
2. Verify that the distiller is disconnected from the electrical
outlet.
3. Remove the condenser. Disconnect the linkage system
for the entry of vapour and the connection which links
the condenser to the distilled product storage tank.
4. Remove screws joining the ventilator with the condenser.
Disconnect the ventilator terminals from its connection
points.
5. Remove the ventilator and clean the dirt accumulated
on the blades. Lubricate the rotation axis with mineral
oil (two drops).
6. Remove the condenser. Aspirate dirt, dust and fuf
accumulated on the surface of the diffusing fins.
Compressed air or a brush dampened with soap and
water can also be used.
7. Rinse the parts.
8. Dry.
9. Assemble again in the reverse order to that described.
Sterilization of the distilled water storage tank
1
The minerals deposited on the cover of the immersion resistors are
particularly poor heat conductors in that they impede an ef cient transfer
of heat between the immersion resistance and the water in the distillation
process. This makes the temperature of the resistance rise above that it
would reach in normal operating conditions, deteriorating its condition and
integrity..
CHAPTER 8 WATER DI STI LLER
56
Frequency: Occasionally
Before operating a new water distiller, it is recommended
to insure that the distilled water storage tank is sterile and
clean. To carry out the sterilization, use a chemical process
with domestic bleach (chlorine based), for example. The
procedure is as follows:
1. Verify that the main switch is of.
2. Open the front panel in order to access the storage tank
for the distilled product.
3. Remove the activated carbon flter from its housing.
4. Prepare a chlorine bleach solution with a concentration
of 200 ppm and add it to the storage tank.
5. Allow the solution to interact with the tank for at least
three hours.
6. Empty the storage tank using the drainage line.
7. Turn on the distiller and allow the storage tank to be
flled with distilled water.
8. Drain the storage tank again.
9. Install the activated carbon flter in its place.
10. Allow the distiller to fll the storage tank with distilled
water. The activated carbon filter will remove any
remnant of chlorine bleach used.
TROUBLESHOOTING TABLE
PROBLEM PROBABLE CAUSE SOLUTION
The distiller does not produce distilled water. There is no energy supply. Verify that the electric connector is well adjusted in
the electrical outlet.
Confrm that there is power in the circuit feeding the
distiller.
Verify that the main switch is in the on position.
Test to ensure that there is water in the vapour
generator or boiling chamber.
The immersion resistance is burnt out. Verify the integrity of the immersion resistance.
Measure electrical continuity or resistance in
ohms. Substitute with another that has the same
characteristics as the original.
There is water around the distiller. The distiller or some of its components are
incorrectly adjusted.
Test the flter to ensure that the activated carbon is
well installed and that water fows through it.
Verify that the collector tank of condensed liquid is
properly placed.
Confrm that the drainage installation does not have
leaks.
There is vapour around the distiller. The distillers ventilation is inadequate. Verify that the distiller has free space around it and
at the back.
Test that there are no objects interfering with the
fow of air towards the distiller.
Remove any object afecting the fow of air
The refrigeration ventilation does not function. Verify the condition of the ventilator. If it is turned
ON and not functioning, substitute the ventilator
with another with the same characteristics as the
original.
The distilled water has a favour. The carbon flter is worn out. Replace the activated carbon flter.
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BASIC DEFINITIONS
Distillation. A process through which a fuid in liquid phase is heated until converted into vapour and then cooled and condensed back into liquid phase. The
distillation process is used for separating mixed substances, taking advantage of their diference in volatility. To obtain very pure substances, consecutive distillation
cycles are performed with the aim of progressively eliminating other substances present in the mix.
Hardness (of water). A chemical characteristic of water determined by the carbonate, bicarbonate, chlorine, sulphate and occasionally calcium nitrate and
magnesium content. The resulting resistance is undesirable in some processes. There are two types of resistors in water.
Temporary hardness. This is determined by the magnesium and calcium carbonate and bicarbonate content. It may be eliminated by boiling the water and
subsequently fltering out the precipitate. It is also known as carbonate resistance.
Permanent hardness. This is determined by all the calcium and magnesium salts, except the carbonates and bicarbonates. It cannot be eliminated by the
boiling of water and it is also known as non-bicarbonate resistance.
Interpretation of resistance:
Resistance as CaCO
3
interpretation
075 soft water
75150 water with little resistance
150300 resistant water
> 300 water with great resistance
In potable water, the maximum limit allowed is 300 mg /l.
In water for heaters, the limit is 0 mg / l.
Calcium resistance or hardness (RCa
++
). Quantity of calcium present in water.
Magnesium resistance or hardness (RMg
++
). Quantity of magnesium present in water.
Total resistance or general hardness [TH]. Quantity in calcium [Ca] solution and magnesium [Mg] as cations, without taking into account the nature of the
anions present in the water. It is expressed as ppm (parts per million) of calcium carbonate (CaCo
3
).
Incrustation (scale). A name given to solids in suspension deposited in layers on the surface of water storage containers.
Solution. A homogenous mix of two or more substances characterized by the absence of chemical reactions between the components of the liquid mixture. The
liquid component which generally appears in greater proportion is called the solvent and that found in a lesser quantity in solution, the solute.
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Chapter 9
Dilutor
The dilutor is used for diluting substances. Dilute comes
from the Latin word diluere and means to add liquid to a
solution. Solutions are defned as homogeneous mixtures
of two or more components which may be gaseous,
liquid or solid. To dilute is to reduce the strength of a
fuid in a solvent, generally water. The dilutor facilitates
the preparation of liquid mixtures, until these achieve a
proportion (concentration) suitable for use in different
diagnostic processes. The identification of this type of
equipment is generalized using the word dilutor.
DIAGRAM OF A DILUTOR
PURPOSE OF THE DILUTOR
The purpose of the dilutor is to prepare mixtures of
substances to achieve determined concentrations and
volumes as done with a pipette, but with the advantage of
an automated or programmed process. Dilutors vary in size
and complexity. Their capacity depends on the models and
manufacturers. They can control known volumes between
25 l (microlitres) and 25 ml (millilitres).
GMDN Code 15133
ECRI Code 15-133
Denomination
Dilutors
Propeller
Control Panel
Dispenser
Figure 22. Dilutor diagram
CHAPTER 9 DI LUTOR
60
OPERATION PRINCIPLES
The dilutor has various components which interact in a
coordinated manner to handle liquids and mix volumes
with great precision, which allows known solutions of
between 1 l and 25 ml to be prepared. The dilutor has in
general, the following components:
1. A propulsion system
2. A control system
3. A dispensing system
Propulsion system
This is generally constituted of positive displacement
systems as found in syringes. One or more selectable
syringes (with a varying capacity) is/are used in the dilutor
to control the volume to be mixed or diluted. The syringes
pistons are moved by a mechanism which controls their
position. Aspirated volumes or deliveries are calculated by
means of the following equation:
Where:
V = fraction of the volume delivered by the syringe
when the piston has a displacement l.
A = piston area.
The total volume aspirated or delivered is the corresponding
integral:

where l
o
and l
1
correspond to the positions that defne the
pistons displacement.
Controlling how the pistons move facilitates good control
over the volumes handled. The displacement system is
activated by an electric motor which moves a very precise
nuts and screws system and changes the position of the
piston. A set of valves controlling the aspiration and supply
processes complements the syringes and their displacement
systems. The confguration of the dilutor depends on the
model and manufacturers.
Control system
Modern dilutors have a control system which is automatic or
controlled by microprocessors. The latter allow the following
to be selected and controlled:
1. Mixing processes and/or dissolution of substances
(programmable)
2. Predefned volume supply
3. Supply or suction velocities
4. Number of required cycles
5. Size or volume of selected syringes
6. Time
7. Priming and cleaning cycles
8. Quality control procedures
In order to give a clearer idea of the technical complexity
achieved, a diagram of the control system based on a
microprocessor displaying some of the dilutor functions is
shown next. The controls for this type of device are generally
symmetrical if they control two injectors.
V = Al
V =
l

V = A l
l
o
l
1

Left Injector Screen


Increase, Decrease
Parameter Controls
Operation Mode Controls
Right Injector Velocity Control
Right Injector Screen
Right Syringe Size
Volume Control
Right Syringe Selector Size
Main Switch
Figure 23. Dilutor controls
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Dispenser system
The dispenser system is composed of a set of high precision
syringes and devices called dispensers, through which
fuids are supplied according to their volumes and selected
velocities. These syringes are selected and installed in the
dilutor depending on the densities, viscosities, and volumes
of fluids to be manipulated. The fluids are transported
through flexible tubes, whose diameters, lengths and
chemical compatibility are taken into account in the design
and manufacturing process for suitability with the selected
activity. These tubes are linked using connections manually
adjustable. Normally, the syringes are classifed according to
their use (e.g. syringes for reagents, diluents, samples), and
the volume these manipulate. The following table shows an
example of how they are classifed according to their size
and managed volumes.
Opposite, the components of the dispensing system (syringe
and dispenser) are shown.
INSTALLATION REQUIREMENTS
The dilutor must be installed on a clean, dry and extremely
levelled counter or work surface, far from areas where there
may be vapours which can afect its functioning.
There must be free space around the equipment for
facilitating ventilation and the passage of cables and
interconnection lines and cables with the solvent containers,
computers or supply systems. The space around the dilutor
should be approximately 10 cm.
There must be a 115 V, 60 Hz electrical outlet in good
condition with a ground pole or alternatively one of 220240
V, 50/60 Hz, depending on the manufacturers specifcations
and/or the electrical norms in the country of use.
ROUTINE MAINTENANCE
The routine maintenance focuses mainly on eliminating
contaminants which may accumulate inside the fluid
mechanisms and/or lines. The most common routines are
the following:
Cleaning of exterior surfaces
Frequency: Daily
Warning: Disconnect the dilutor from the electrical feed
outlet before beginning the external cleaning process.
1. Clean the exterior surfaces using a clean piece of cloth
dampened with a mild detergent mixed with water.
2. Lightly rub the surfaces of the dilutor and the
accessories.
3. Dry the treated surfaces.
Warning: Avoid humidity from entering the compartment
of the electrical and electronic components.
Part No.
(Depending
on the
manufacturer)
Model
(Depending
on the
manufacturer)
Syringe size
Range
(Processed
volume)
Duct size
1
Aqueous
solution
Viscous liquids
DM DM 25 l 2.525 l 18 18
DM DM 50 l 550 l 18 18
DM DM 100 l 10100 l 18 18
DM DM 250 l 25250 l 18 18
DM DM 500 l 50500 l 18 18
DM DM 1 ml 1001 000 l 18 18
DM DM 2.5 ml 2502 500 l 18 12
DM DM 5 ml 5005 000 l 12 12
DM DM 10 ml 1 00010 000 l 12 12
DM DM 25 ml 2 50025 000 l 12 12
Table of syringe size/volumes managed
1
Table 2.4, Microlab 501A, 503A, 504A, Users Manual, Hamilton Company.
Syringe
Dispenser
Figure 24. Syringe and dispenser
CHAPTER 9 DI LUTOR
62
Cleaning of syringes, hoses or lines

Warning: If the dilutor has been in contact with dangerous
substances, the safety and prevention procedures
implemented in the laboratory must be respected.
Frequency: Daily
1. Feed the system with a cleaning solution. Consult the
manufacturer to enquire about the solution to use. Verify
that each systems elements come into contact with the
solution and that air bubbles have been eliminated. This
process is known as priming. In order to feed the system,
the dilutor is connected to a container in which the used
solution is present. Once the priming is complete; the
waste solution goes into another container for fnal
disposal.
2. Clean the system. In order to carry out cleaning, a fuid
which complements the cleaning solution is circulated
(consult the manufacturers recommendations). It is
common to use deionised water as a cleaning fuid.
Depending on the substances processed in the dilutor,
other cleaning agents can be used such as ethanol, urea,
or a 10% bleach solution in deionised water.
Cleaning of the uid conduction system
Frequency: Before putting into service for the rst time
1. Prepare a container with cleaning solution and place
the flling tube inside (manufacturers recommend using
cleaning agents compatible with the dilutor).
2. Place the waste line inside the waste container.
3. Run a feed or priming cycle until the fuids lines becomes
clean.
4. Remove the flling tube from the cleaning solution and
place it inside a container with deionised water. Start a
feed or priming cycle again until the fuid trajectory is
free of cleaning solution. Discard the fuid and rinse the
waste container.
5. Suspend the feed cycle.
6. Place the fuid propulsion system in the rest position.
7. Use the system as it is clean and ready.
Procedure for storing the dilutor
Frequency: Whenever stored for a prolonged period of
time
1. Purge and prime the system using methanol (facilitates
drying).
2. Remove the tubes and syringes.
3. Store the syringes in their original protective covers.
4. Cover the body of the dilutor in order to protect it from
dust.
5. Store.
Quality control
The quality control of dilutors is similar to that of pipettes.
In order to resolve uncertainties, please see the explanation
regarding how calibration is conducted in Chapter 16 on
pipettes.
MAI NT E NANC E MANUAL F OR L ABOR ATORY E QUI P ME NT
63
TROUBLESHOOTING TABLE
PROBLEM PROBABLE CAUSE SOLUTION
The dilutor does not turn on. There is a fault in the electrical feed. Check the electrical connection.
The electrical feed is disconnected. Connect electrical feed cable.
The protection fuse is open. Check the protection fuse. Substitute with an
equivalent one if it is burnt.
The dilutor operates well, but there are no messages
or indications on the screen.
There is possible damage to the LCD screen or in the
emission diodes of the LED light.
Verify that the control is well connected to the
propulsion system.
Call the manufacturers service technician.
The control keys do not function. The dilutor is on the Pause mode. Press the start/end button to complete the path of
the piston.
The dilute is obstructed. There is an internal error. Press the start/end button to complete the path of
the piston and to restart the cycle.
Call the manufacturers service technician, if the fault
persists.
The dilutor does not aspirate nor dispense. The hydraulic systems tubes are defective or
blocked.
Verify that tubes, syringes and connectors are free
from blockages. Clean or substitute.
Incorrect connection of tubes and syringes Test that the tubes, joints, connections and syringes
used are well adjusted.
The propulsion system is defective. Call the manufacturers service technician.
The valves are defective. Remove the valves. Verify that their seals are clean
and reinstall. Substitute for an equivalent valve if
necessary.
The dilutor does not produce precise results. There is air in the fuid circuit. Verify that the feeding tubes are completely
submerged inside the containers which contain the
reagents.
Confrm that the diferent connectors are adjusted.
Verify that the syringes are correctly installed and
there are no leaks.
Test to ensure that the tubes or valves have no leaks.
Reduce the operational speed of the syringe to
eliminate cavitation problems.
The delivery tube is incorrectly selected for the
syringes capacity.
Verify the recommended size of the tube used and its
connections. For small volumes, use the dimensions
recommended by the manufacturer.
A small air gap appears on the tip of the probe after
the fnal aspiration.
The aspiration tube is dirty. Change or clean the aspiration tube.
The aspiration mode is incorrect. Reduce the aspiration speed.
Air is persistently present or there are constant leaks
in the fuid trajectory.
Cavitations are present in the system. The aspiration
speed is very high.
Reduce the propulsion systems speed. Remember
that the more viscous the fuids, the lower the speed
must be used to manipulate them.
The connections are loose, worn out or defective. Adjust the connections by hand. Substitute to tubes
with dimensions corresponding with the fuids
processed.
The piston is defective or the syringe is damaged. Replace the piston or the syringe.
There is a defective valve. Replace the valve.
The dilutor is heating. There is inadequate ventilation. Check the ventilation.
The room temperature is too high. Check the air conditioning system in the area.
The work cycle is very intense. Use the dilutor with less intensity.
CHAPTER 9 DI LUTOR
64
BASIC DEFINITIONS
Cavitations. A phenomenon in fuids when a vacuum is created upon emptying a vessel. The pressure decreases until it reaches the vapour pressure of the fuid.
This produces diverse phenomena such as vaporization of gases dissolved in the liquid or, in the case of water, the formation of vapour bubbles collapsing after an
infnitesimal time lapse, perforating the surfaces of conducts in the immediate vicinity. This occurs in dilutors when using large capacity syringes with elevated
propulsion speed.
Concentration. A quantity measurement of a chemical substance present in a solution. The concept is expressed as the quantity of a substance dissolved into a
solvent. Concentration is expressed in diverse forms; the most common are: molarity [M], molality [m], normality [N], percentage rate of solute.
Dilution. To reduce the concentration of a solution by adding other fuids. The fuid added is known as the diluent. Adding the molecules of a liquid substance with
the molecules of another liquid substance. In order to determine the volume V1 of liquid needed to obtain V2 volume at a concentration C2 from a stock solution of
concentration C1, the following equation is used:

Dispenser. A device used for distributing liquids.
Dispensing. Distributing a fuid at a constant volume or in a progressive form.
Dissolution. Process by which a chemical in solid form is dissolved in a solvent (e.g. water or other liquid). The chemical now in solution is called the solute.
Equivalent gram [Eq]. Mass in grams of solute divided by its equivalent weight [EW]:
Equivalent weight [EW] (of one substance). Results from dividing the molecular weight [MW] by its valency.
Molality [m]. Number of moles of a given substance, for every 1000 g of solvent. Thus an m molal solution is obtained by adding m moles of the substance to
1000 g of water.
Molarity [M] (of a solution component). Number of moles of solute for each litre of fnal solution. A solution n Molar of a salt is obtained by adding n moles from
that salt to water until obtaining one (1) litre of solution. Normally, the formula employed is the following:
Mole. Molecular weight (MW) of the solute expressed in grams:
Normality [N] (of a solute). Number of moles of solute per litre of fnal solution.
Solution. A homogeneous liquid mixture of two or more substances. The dissolved chemical(s) called the solute(s) usually name the solution. The substance in
which the solute(s) are now dissolved is called the solvent. There is a usually greater quantity of solvent than solute(s) in a solution.
Weight/Volume. Relationship in clinical biochemistry expressing the mass of the solution in grams or its submultiples per volume unit in litres or submultiples
of a litre. For example: g/l, mg/ml.
Note: Another type of notation known as part per unitis used for measuring extremely low concentrations. For example: parts per million (ppm) means that there
is a particle of a given substance for each 999 999 particles of other substances.
V
1
=
V
2
C
2
C
1
Eq =
mass(g)
EW (g)
EW =
MW (g)
valency
M =
moles
Vol(L)
moles=
mass(g)
EW
N =
Eq
Vol(L)

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